Antagonists of lysophosphatidic acid receptors

ABSTRACT

Described herein are compounds that are antagonists of lysophosphatidic receptor(s). Also described are pharmaceutical compositions and medicaments that include the compounds described herein, as well as methods of using such antagonists, alone and in combination with other compounds, for treating LPA-dependent or LPA-mediated conditions or diseases.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/122,568, entitled “ANTAGONISTS OF LYSOPHOSPHATIDIC ACID RECEPTORS” filed on Dec. 15, 2008, which is herein incorporated by reference.

FIELD OF THE INVENTION

Described herein are compounds, methods of making such compounds, pharmaceutical compositions and medicaments comprising such compounds, and methods of using such compounds to treat, prevent or diagnose diseases, disorders or conditions associated with one or more of the lysophosphatidic acid (LPA) receptors.

BACKGROUND OF THE INVENTION

Lysophospholipids are membrane-derived bioactive lipid mediators. Lysophospholipids affect fundamental cellular functions that include proliferation, differentiation, survival, migration, adhesion, invasion, and morphogensis. These functions influence many biological processes that include, but are not limited to, neurogensis, angiogenesis, wound healing, fibrosis, immunity, and carcinogenesis.

Lysophosphatidic acid (LPA) is a lysophospholipid that has been shown to act through sets of specific G protein-coupled receptors (GPCRs) in an autocrine and paracrine fashion. LPA binding to its cognate GPCRs (LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, LPA₆) activates intracellular signaling pathways to produce a variety of biological responses. Antagonists of the LPA receptors find use in the treatment of diseases, disorders or conditions in which LPA plays a role.

SUMMARY OF THE INVENTION

In one aspect, presented herein are compounds of Formula (I) that inhibit the physiological activity of lysophosphatidic acid (LPA), and therefore, are useful as agents for the treatment or prevention of diseases in which inhibition of the physiological activity of LPA is useful, such as diseases in which an LPA receptor participates, is involved in the etiology or pathology of the disease, or is otherwise associated with at least one symptom of the disease. In a related aspect, such compounds are useful as agents for the treatment or prevention of side effects, complications, or adverse events associated with the use of a different therapeutic agent or therapeutic action (e.g., radiation, surgery, etc) used in treating a disease or condition.

In one aspect, the compounds of Formula (I) are useful for the treatment of fibrosis of organs (liver, kidney, lung, heart and the like), liver diseases (acute hepatatis, chronic hepatitis, liver fibrosis, liver cirrhosis, portal hypertension, regenerative failure, non-alcoholic steatohepatitis (NASH), liver hypofunction, hepatic blood flow disorder, and the like), cell proliferative disease (cancer (solid tumor, solid tumor metastasis, vascular fibroma, myeloma, multiple myeloma, Kaposi's sarcoma, leukemia, chronic lymphocytic leukemia (CLL) and the like) and invasive metastasis of cancer cell, and the like), inflammatory disease (psoriasis, nephropathy, pneumonia and the like), gastrointestinal tract disease (irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), abnormal pancreatic secretion, and the like), renal disease, urinary tract-associated disease (benign prostatic hyperplasia or symptoms associated with neuropathic bladder disease, spinal cord tumor, hernia of intervertebral disk, spinal canal stenosis, symptoms derived from diabetes, lower urinary tract disease (obstruction of lower urinary tract, and the like), inflammatory disease of lower urinary tract, dysuria, frequent urination, and the like), pancreas disease, abnormal angiogenesis-associated disease (arterial obstruction and the like), scleroderma, brain-associated disease (cerebral infarction, cerebral hemorrhage, and the like), neuropathic pain, peripheral neuropathy, and the like, ocular disease (age-related macular degeneration (AMD), diabetic retinopathy, proliferative vitreoretinopathy (PVR), cicatricial pemphigoid, glaucoma filtration surgery scarring, and the like). In one aspect, the compounds of Formula (I) are used in the treatment of fibrotic diseases or conditions.

In one aspect, described herein are compounds of Formula (I), pharmaceutically acceptable salts, solvates, and prodrugs thereof. Compounds of Formula (I) are antagonists of at least one of the LPA receptors selected from LPA₁, LPA₂, LPA₃, LPA₄, LPA₅ and LPA₆. In one embodiment, compounds of Formula (I) are antagonists of LPA₁. In one embodiment, compounds of Formula (I) are antagonists of LPA₁ and/or LPA₃. In some embodiments, compounds of Formula (I) are antagonists of LPA₁ and/or LPA₂. In some embodiments, compounds of Formula (I) are selective antagonists for one of the LPA receptors relative to the other LPA receptors. In some embodiments, such a selective antagonist is selective for the LPA₁ receptor. In some embodiments, such a selective antagonist is selective for the LPA₂ receptor. In some embodiments, such a selective antagonist is selective for the LPA₃ receptor.

Compounds of Formula (I) are used in the treatment of diseases, disorders, or conditions in which activation of at least one LPA receptor by LPA contributes to the symptomology or progression of the disease, disorder or condition. In one aspect, the methods, compounds, pharmaceutical compositions, and medicaments described herein comprise antagonists of LPA receptors. In one aspect, the methods, compounds, pharmaceutical compositions, and medicaments described herein comprise antagonists of LPA₁, LPA₂, or LPA₃, or combinations thereof.

In one aspect, provided herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein

-   -   R¹ is —CO₂H, —CO₂R^(D), —CN, tetrazolyl, —C(═O)NH₂, —C(═O)NHR¹⁰,         —C(═O)NHSO₂R¹⁰ or —C(═O)NHCH₂CH₂SO₃H; R^(D) is H or C₁-C₄alkyl;     -   L¹ is absent or C₁-C₆alkylene;     -   R³ is H, C₁-C₄alkyl, C₃-C₆cycloalkyl, or C₁-C₄-fluoroalkyl;     -   R⁷ is H or C₁-C₄alkyl;     -   R⁸ is H, C₁-C₄alkyl, or C₁-C₄-fluoroalkyl;     -   R¹⁰ is a C₁-C₆alkyl, C₁-C₆-fluoroalkyl, C₃-C₆cycloalkyl, or a         substituted or unsubstituted phenyl;     -   each of R^(A), R^(B), and R^(C) are independently selected from         H, F, Cl, Br, I, —CN, —OH, C₁-C₄alkyl, C₁-C₄-fluoroalkyl,         C₁-C₄-fluoroalkoxy, C₁-C₄alkoxy, and C₁-C₄heteroalkyl;     -   m is 0, 1, or 2; n is 0, 1, or 2; p is 0, 1, or 2.

In one aspect, provided is are compounds presented in Table 1, Table 2, Table 3, FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, and FIG. 7.

Compounds of Formula (I) are antagonists of at least one LPA receptor. In some embodiments, the compound of Formula (I) is an antagonist of LPA₁. In some embodiments, the compound of Formula (I) is an antagonist of LPA₂. In some embodiments, the compound of Formula (I) is an antagonist of LPA₃.

In some embodiments, presented herein are compounds selected from active metabolites, tautomers, solvates, pharmaceutically acceptable salts or prodrugs of a compound of Formula (I).

In some embodiments, provided is a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I). In some embodiments, the pharmaceutical composition also contains at least one pharmaceutically acceptable inactive ingredient.

In some embodiments, provided is a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable inactive ingredient. In one aspect, the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, oral administration, inhalation, nasal administration, topical administration, ophthalmic administration or otic administration. In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.

In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutically active agents selected from: corticosteroids, immunosuppresants, analgesics, anti-cancer agent, anti-inflammatories, chemokine receptor antagonists, bronchodilators, leukotriene receptor antagonists, leukotriene formation inhibitors, monoacylglycerol kinase inhibitors, phospholipase A₁ inhibitors, phospholipase A₂ inhibitors, and lysophospholipase D (lysoPLD) inhibitors, autotaxin inhibitors, decongestants, antihistamines, mucolytics, anticholinergics, antitussives, expectorants, and β-2 agonists.

In some embodiments, provided is a method comprising administering a compound of Formula (I) to a human with a LPA-dependent or LPA-mediated disease or condition. In some embodiments, the human is already being administered one or more additional therapeutically active agents other than a compound of Formula (I). In some embodiments, the method further comprises administering one or more additional therapeutically active agents other than a compound of Formula (I).

In some embodiments, the one or more additional therapeutically active agents other than a compound of Formula (I) are selected from: corticosteroids, immunosuppresants, analgesics, anti-cancer agent, anti-inflammatories, chemokine receptor antagonists, bronchodilators, leukotriene receptor antagonists, leukotriene formation inhibitors, monoacylglycerol kinase inhibitors, phospholipase A₁ inhibitors, phospholipase A₂ inhibitors, and lysophospholipase D (lysoPLD) inhibitors, autotaxin inhibitors, decongestants, antihistamines, mucolytics, anticholinergics, antitussives, expectorants, and (3-2 agonists.

In another aspect is the use of a compound of Formula (I) in the manufacture of a medicament for treating a disease, disorder or condition in which the activity of at least one LPA receptor contributes to the pathology and/or symptoms of the disease or condition. In one embodiment of this aspect, the LPA receptor is selected from LPA₁, LPA₂, LPA₃, LPA₄, LPA₅ and LPA₆. In some embodiments, the LPA receptor is LPA₁. In some embodiments, the LPA receptor is LPA₂. In some embodiments, the LPA receptor is LPA₃. In some embodiments, the disease or condition is any of the diseases or conditions specified herein.

Also provided is a method of inhibiting the physiological activity of LPA in a mammal comprising administering a therapuetically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to the mammal in need thereof.

In one aspect, provided is a medicament for treating a LPA-dependent or LPA-mediated disease or condition in a mammal comprising a therapeutically effective amount of a compound of Formula (I).

In some cases disclosed herein is the use of a compound of Formula (I) in the manufacture of a medicament for the treatment of a LPA-dependent or LPA-mediated disease or condition.

In some cases disclosed herein is the use of a compound of Formula (I) in the treatment or prevention of a LPA-dependent or LPA-mediated disease or condition.

In one aspect, is a method for treating or preventing a LPA-dependent or LPA-mediated disease or condition in a mammal comprising administering a therapuetically effective amount of a compound of Formula (I).

In one aspect, LPA-dependent or LPA-mediated diseases or conditions include, but are not limited to, fibrosis of organs or tissues, scarring, liver diseases, dermatological conditions, cancer, cardiovascular disease, respiratory diseases or conditions, inflammatory disease, gastrointestinal tract disease, renal disease, urinary tract-associated disease, inflammatory disease of lower urinary tract, dysuria, frequent urination, pancreas disease, arterial obstruction, cerebral infarction, cerebral hemorrhage, pain, peripheral neuropathy, and fibromyalgia.

In some embodiments, the LPA-dependent or LPA-mediated disease or condition is selected from idiopathic pulmonary fibrosis; other diffuse parenchymal lung diseases of different etiologies including iatrogenic drug-induced fibrosis, occupational and/or environmental induced fibrosis, granulomatous diseases (sarcoidosis, hypersensitivity pneumonia), collagen vascular disease, alveolar proteinosis, langerhans cell granulomatosis, lymphangioleiomyomatosis, inherited diseases (Hermansky-Pudlak Syndrome, tuberous sclerosis, neurofibromatosis, metabolic storage disorders, familial interstitial lung disease); radiation induced fibrosis; chronic obstructive pulmonary disease (COPD); scleroderma; bleomycin induced pulmonary fibrosis; chronic asthma; silicosis; asbestos induced pulmonary fibrosis; acute respiratory distress syndrome (ARDS); kidney fibrosis; tubulointerstitium fibrosis; glomerular nephritis; focal segmental glomerular sclerosis; IgA nephropathy; hypertension; Alport; gut fibrosis; liver fibrosis; cirrhosis; alcohol induced liver fibrosis; toxic/drug induced liver fibrosis; hemochromatosis; nonalcoholic steatohepatitis (NASH); biliary duct injury; primary biliary cirrhosis; infection induced liver fibrosis; viral induced liver fibrosis; and autoimmune hepatitis; corneal scarring; hypertrophic scarring; Duputren disease, keloids, cutaneous fibrosis; cutaneous scleroderma; spinal cord injury/fibrosis; myelofibrosis; vascular restenosis; atherosclerosis; arteriosclerosis; Wegener's granulomatosis; Peyronie's disease, chronic lymphocytic leukemia, tumor metastasis, transplant organ rejection, endometreosis, neonatal respiratory distress syndrome and neuropathic pain.

In one aspect, provided is a method for the treatment or prevention of organ fibrosis in a mammal comprising administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

In some embodiments, the organ fibrosis comprises lung fibrosis, renal fibrosis, or hepatic fibrosis.

In one aspect, provided is a method of improving lung function in a mammal comprising administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to the mammal in need thereof. In one aspect, the mammal has been diagnosed as having lung fibrosis.

In one aspect, compounds disclosed herein are used to treat idiopathic pulmonary fibrosis (usual interstitial pneumonia) in a mammal.

In some embodiments, compounds disclosed herein are used to treat diffuse parenchymal interstitial lung diseases in mammal: iatrogenic drug induced, occupational/environmental (Farmer lung), granulomatous diseases (sarcoidosis, hypersensitivity pneumonia), collagen vascular disease (scleroderma and others), alveolar proteinosis, langerhans cell granulonmatosis, lymphangioleiomyomatosis, Hermansky-Pudlak Syndrome, Tuberous sclerosis, neurofibromatosis, metabolic storage disorders, familial interstitial lung disease.

In some embodiments, compounds disclosed herein are used to treat post-transplant fibrosis associated with chronic rejection in a mammal. Bronchiolitis obliterans for lung transplant.

In some embodiments, compounds disclosed herein are used to treat cutaneous fibrosis in a mammal: cutaneous scleroderma, Dupuytren disease, keloids.

In one aspect, compounds disclosed herein are used to treat hepatic fibrosis with or without cirrhosis in a mammal: toxic/drug induced (hemochromatosis), alcoholic liver disease, viral hepatitis (hepatitis B virus, hepatitis C virus, HCV), nonalcoholic liver disease (NASH), metabolic and auto-immune.

In one aspect, compounds disclosed herein are used to treat renal fibrosis in a mammal: tubulointerstitium fibrosis, glomerular sclerosis.

In any of the aforementioned aspects involving the treatment of LPA dependent diseases or conditions are further embodiments comprising administering at least one additional agent in addition to the administartion of a compound having the structure of Formula (I). In various embodiments, each agent is administered in any order, including simultaneously.

In any of the embodiments disclosed herein, the mammal is a human.

In some embodiments, compounds provided herein are administered to a human. In some embodiments, compounds provided herein are orally administered to a human.

In some embodiments, compounds provided herein are used as antagonists of at least one LPA receptor. In some embodiments, compounds provided herein are used for inhibiting the activity of at least one LPA receptor or for the treatment of a disease or condition that would benefit from inhibition of the activity of at least one LPA receptor. In one aspect, the LPA receptor is LPA₁.

In other embodiments, compounds provided herein are used for the formulation of a medicament for the inhibition of LPA₁ activity.

Other objects, features and advantages of the compounds, methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the instant disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustrative examples of compounds described herein.

FIG. 2. Illustrative examples of compounds described herein.

FIG. 3. Illustrative examples of compounds described herein.

FIG. 4. Illustrative examples of compounds described herein.

FIG. 5. Illustrative examples of compounds described herein.

FIG. 6. Illustrative examples of compounds described herein.

FIG. 7. Illustrative examples of compounds described herein.

DETAILED DESCRIPTION OF THE INVENTION

Lysophospholipids (such as lysophosphatidic acid (LPA)) affect fundamental cellular functions that include cellular proliferation, differentiation, survival, migration, adhesion, invasion, and morphogensis. These functions influence many biological processes that include neurogensis, angiogenesis, wound healing, immunity, and carcinogenesis.

LPA acts through sets of specific G protein-coupled receptors (GPCRs) in an autocrine and paracrine fashion. LPA binding to its cognate GPCRs (LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, LPA₆) activates intracellular signaling pathways to produce a variety of biological responses.

LPA has a role as a biological effector molecule, and has a diverse range of physiological actions such as, but not limited to, effects on blood pressure, platelet activation, and smooth muscle contraction, and a variety of cellular effects, which include cell growth, cell rounding, neurite retraction, and actin stress fiber formation and cell migration. The effects of LPA are predominantly receptor mediated.

Activation of the LPA receptors (LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, LPA₆) with LPA mediates a range of downstream signaling cascades. The actual pathway and realized end point are dependent on a range of variables that include receptor usage, cell type, expression level of a receptor or signaling protein, and LPA concentration. Nearly all mammalian cells, tissues and organs co-express several LPA-receptor subtypes, which indicates that LPA receptors signal in a cooperative manner LPA₁, LPA₂, and LPA₃ share high amino acid sequence similarity.

Illustrative Biological Activity

LPA regulates many important functions of fibroblasts in wound healing, including proliferation, migration, differentiation and contraction. Fibroblast proliferation is required in wound healing in order to fill an open wound. In contrast, fibrosis is characterized by intense proliferation and accumulation of myofibroblasts that actively synthesize ECM and proinflammatory cytokines. LPA can either increase or suppress the proliferation of cell types important in wound healing.

Tissue injury initiates a complex series of host wound-healing responses; if successful, these responses restore normal tissue structure and function. If not, these responses can lead to tissue fibrosis and loss of function.

A number of muscular dystrophies are characterized by a progressive weakness and wasting of musculature, and by extensive fibrosis. It has been shown that LPA treatment of cultured myoblasts induced significant expression of connective tissue growth factor (CTGF). CTGF subsequently induces collagen, fibronectin and integrin expression and induces dedifferentiation of these myoblasts. Treatment of a variety of cell types with LPA induces reproducible and high level induction of CTGF. CTGF is a profibrotic cytokine, signaling down-stream and in parallel with TGFβ.

LPA and LPA₁ play key pathogenic roles in pulmonary fibrosis. Fibroblast chemoattractant activity plays an important role in the lungs in patients with pulmonary fibrosis. Profibrotic effects of LPA₁-receptor stimulation is explained by LPA₁-receptor-mediated vascular leakage and increased fibroblast recruitment, both profibrotic events. The LPA-LPA₁ pathway has a role in mediating fibroblast migration and vascular leakage in IPF. The end result is the aberrant healing process that characterises this fibrotic condition.

The LPA-LPA2 pathway contributes to the activation of the TGF-β pathway in pulmonary fibrosis. In some embodiments, compounds that inhibit LPA2 show efficacy in the treatment of lung fibrosis. In some embodiments, compounds that inhibit both LPA1 and LPA2 show improved efficacy in the treatment of lung fibrosis compared to compounds which inhibit only LPA1 or LPA2.

LPA and LPA₁ are involved in the etiology of kidney fibrosis. In mice invalidated for the LPA₁ receptor (LPA₁ (−/−), the development of renal fibrosis was significantly attenuated. Unilateral ureteral obstruction (UUO; animal model of renal fibrosis) mice treated with the LPA receptor antagonist Ki16425 closely resembled the LPA₁ (−/−) mice.

LPA is implicated in liver disease and fibrosis. Plasma LPA levels and serum autotoxin are elevated in hepatitis patients and animal models of liver injury in correlation with increased fibrosis. LPA also regulates liver cell function. LPA₁ and LPA₂ receptors are expressed by mouse hepatic stellate cells and LPA stimulates migration of hepatic myofibroblasts.

LPA is in involved in wound healing in the eye. LPA₁ and LPA₃ receptors are detectable in the normal rabbit corneal epithelial cells, keratocytes and endothelial cells and LPA₁ and LPA₃ expression are increased in corneal epithelial cells following injury.

LPA is present in the aqueous humor and the lacrimal gland fluid of the rabbit eye and these levels are increased in a rabbit corneal injury model.

LPA induces actin stress fiber formation in rabbit corneal endothelial and epithelial cells and promotes contraction corneal fibroblasts. LPA also stimulates proliferation of human retinal pigmented epithelial cells.

LPA is implicated in myocardial infarction and cardiac fibrosis. Serum LPA levels are increased in patients following mycocardial infarction (MI) and LPA stimulates proliferation and collagen production (fibrosis) by rat cardiac fibroblasts. Both LPA1 and LPA3 receptors are highly expressed in human heart tissue.

In one aspect, compounds of Formula (I) are used to treat or prevent fibrosis in a mammal. In one aspect, compounds of Formula (I) are used to treat or prevent fibrosis of an organ or tissue in a mammal.

The terms “fibrosis” or “fibrosing disorder,” as used herein, refers to conditions that are associated with the abnormal accumulation of cells and/or fibronectin and/or collagen and/or increased fibroblast recruitment and include but are not limited to fibrosis of individual organs or tissues such as the heart, kidney, liver, joints, lung, pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletal and digestive tract.

Exemplary diseases, disorders, or conditions that involve fibrosis include, but are not limited to: Lung diseases associated with fibrosis, e.g., idiopathic pulmonary fibrosis, pulmonary fibrosis secondary to systemic inflammatory disease such as rheumatoid arthritis, scleroderma, lupus, cryptogenic fibrosing alveolitis, radiation induced fibrosis, chronic obstructive pulmonary disease (COPD), scleroderma, chronic asthma, silicosis, asbestos induced pulmonary or pleural fibrosis, acute lung injury and acute respiratory distress (including bacterial pneumonia induced, trauma induced, viral pneumonia induced, ventilator induced, non-pulmonary sepsis induced, and aspiration induced); Chronic nephropathies associated with injury/fibrosis (kidney fibrosis), e.g., glomerulonephritis secondary to systemic inflammatory diseases such as lupus and scleroderma, diabetes, glomerular nephritis, focal segmental glomerular sclerosis, IgA nephropathy, hypertension, allograft and Alport; Gut fibrosis, e.g., scleroderma, and radiation induced gut fibrosis; Liver fibrosis, e.g., cirrhosis, alcohol induced liver fibrosis, nonalcoholic steatohepatitis (NASH), biliary duct injury, primary biliary cirrhosis, infection or viral induced liver fibrosis (e.g., chronic HCV infection), and autoimmune hepatitis; Head and neck fibrosis, e.g., radiation induced; Corneal scarring, e.g., LASIK (laser-assisted in situ keratomileusis), corneal transplant, and trabeculectomy; Hypertrophic scarring and keloids, e.g., burn induced or surgical; and Other fibrotic diseases, e.g., sarcoidosis, scleroderma, spinal cord injury/fibrosis, myelofibrosis, vascular restenosis, atherosclerosis, arteriosclerosis, Wegener's granulomatosis, mixed connective tissue disease, and Peyronie's disease.

In one aspect, a mammal suffering from one of the following non-limiting exemplary diseases, disorders, or conditions will benefit from therapy with a compound of Formula (I): atherosclerosis, thrombosis, heart disease, vasculitis, formation of scar tissue, restenosis, phlobitis, COPD (chronic obstructive pulmonary disease), pulmonary hypertension, pulmonary fibrosis, pulmonary inflammation, bowel adhesions, bladder fibrosis and cystitis, fibrosis of the nasal passages, sinusitis, inflammation mediated by neutrophils, and fibrosis mediated by fibroblasts.

In one aspect, compounds of Formula (I) are used to treat a dermatological disorders in a mammal Dermatological disorders include, but are not limited to, proliferative or inflammatory disorders of the skin such as, atopic dermatitis, bullous disorders, collagenoses, psoriasis, psoriatic lesions, dermatitis, contact dermatitis, eczema, urticaria, rosacea, wound healing, scarring, hypertrophic scarring, keloids, Kawasaki Disease, rosacea, Sjogren-Larsso Syndrome, urticaria.

In some embodiments, provided is a method of reducing lung injury, vascular leakage, inflammation and/or fibrosis in a mammal comprising administering to the mammal a selective LPA1 receptor antagonist. In some embodiments, provided is a method of reducing lung injury, vascular leakage, inflammation and fibrosis in a mammal comprising administering to the mammal a selective LPA1 receptor antagonist. In some embodiments, provided is a method of attenuating fibrosis in a mammal comprising administering a selective LPA1 receptor antagonist. In some embodiments, provided is a method of attenuating tissue remodeling and fibrosis in a mammal comprising administering a selective LPA1 receptor antagonist.

In some embodiments, provided is a method of decreasing cytokine production in a mammal comprising administering a selective LPA1 receptor antagonist. In some embodiments, the method of decreasing cytokine production in a mammal comprising administering a selective LPA1 receptor antagonist results in a reduction of tissue damage and fibrosis in a mammal.

In some embodiments, provided is a method of treating fibrosis is a mammal comprising administering to the mammal a selective LPA1 receptor antagonist. In some embodiments, provided is a method of treating fibrosis in a mammal while maintaining body weight in the mammal comprising administering to the mammal a selective LPA1 receptor antagonist. In some embodiments, provided is a method of treating respiratory disease in a mammal comprising administering to the mammal a selective LPA1 receptor antagonist.

In some embodiments, provided is a method of treating fibrosis in a mammal with a selective LPA1 receptor anatgonist, wherein the fibrosis in the mammal is not responsive to treatment with pirfenidone. In some embodiments, the LPA1 receptor antagonist is a compound of Formula (I).

As shown in the Examples, a selective LPA1 receptor antagonist reduced lung fibrosis, kidney fibrosis and liver fibrosis in various animal models of fibrosis.

LPA is released following tissue injury. LPA₁ plays a role in the initiation of neuropathic pain. In one aspect, compounds of Formula (I) are used in the treatment of pain in a mammal. In one aspect, the pain is acute pain or chronic pain. In another aspect, the pain is neuropathic pain. In another aspect, the pain is cancer pain. In one aspect, compounds of Formula (I) are used in the treatment of fibromylagia.

Lysophospholipid receptor signaling plays a role in the etiology of cancer. Lysophosphatidic acid (LPA) and its G protein-coupled receptors (GPCRs) LPA₁, LPA₂, and/or LPA₃ play a role in the development of several types of cancers.

LPA contributes to tumorigenesis by increasing motility and invasiveness of cells. LPA has been implicated in the initiation or progression of ovarian cancer. LPA is present at significant concentrations (2-80 μM) in the ascitic fluid of ovarian cancer patients. LPA receptors (LPA2 and LPA3) are also overexpressed in ovarian cancer cells as compared to normal ovarian surface epithelial cells. LPA has also been implicated in the initiation or progression of prostate cancer, breast cancer, melanoma, head and neck cancer, bowel cancer (colorectal cancer), thyroid cancer, glioblastoma, and other cancers.

LPA receptors mediate both migration of and invasion by pancreatic cancer cell lines: Ki16425 and LPA₁-specific siRNA effectively blocked in vitro migration in response to LPA and peritoneal fluid (ascites) from pancreatic cancer patients; in addition, Ki16425 blocked the LPA-induced and ascites-induced invasion activity of a highly peritoneal metastatic pancreatic cancer cell line (Yamada et al, J. Biol. Chem., 279, 6595-6605, 2004).

Colorectal carcinoma cell lines show significant expression of LPA₁ mRNA and respond to LPA by cell migration and production of angiogenic factors. Overexpression of LPA receptors has a role in the pathogenesis of thyroid cancer. LPA₃ was originally cloned from prostate cancer cells, concordant with the ability of LPA to induce autocrine proliferation of prostate cancer cells.

LPA has stimulatory roles in cancer progression in many types of cancer. LPA is produced from and induces proliferation of prostate cancer cell lines. LPA induces human colon carcinoma DLD1 cell proliferation, migration, adhesion, and secretion of angiogenic factors through LPA₁ signalling. In other human colon carcinoma cells lines (HT29 and WiDR), LPA enhances cell proliferation and secretion of angiogenic factors. In other colon cancer cell lines, LPA₂ and LPA₃ receptor activation results in proliferation of the cells. LPA₁ is implicated in bone metastasis and Ki16425 has been shown to inhibit metastasis to bone in vivo (Boucharaba et al., Proc. Natl. Acad. Sci. USA, 103, 9643-9648, 2006).

In one aspect, a compound of Formula (I) is used in the treatment of cancer. In one aspect, compounds of Formula (I) are used in the treatment of malignant and benign proliferative disease. In one aspect, compounds of Formula (I) are used to prevent or reduce proliferation of tumor cells, invasion and metastasis of carcinomas, pleural mesothelioma or peritoneal mesothelioma, cancer pain, bone metastases. In one aspect is a method of treating cancer in a mammal, the method comprising administering to the mammal a compound of Formula (I) and a second therapeutic agent, wherein the second therapeutic agent is an anti-cancer agent. In some embodiments, radiation therapy is also used.

The types of cancer include, but is not limited to, solid tumors (such as those of the bladder, bowel, brain, breast, endometrium, heart, kidney, lung, lymphatic tissue (lymphoma), ovary, pancreas or other endocrine organ (thyroid), prostate, skin (melanoma or basal cell cancer) or hematological tumors (such as the leukemias) at any stage of the disease with or without metastases.

In one aspect, LPA is a contributor to the pathogenesis of respiratory diseases. Proinflammatory effects of LPA include degranulation of mast cells, contraction of smooth-muscle cells and release of cytokines from dendritic cells. LPA induces the secretion of IL-8 from human bronchial epithelial cells. IL-8 is found in increased concentrations in BAL fluids from patients with asthma, chronic obstructive lung disease, pulmonary sarcoidosis and acute respiratory distress syndrome and 11-8 has been shown to exacerbate airway inflammation and airway remodeling of asthmatics. LPA1, LPA2 and LPA3 receptors have all been shown to contribute to the LPA-induced IL-8 production.

Administration of LPA in vivo induces airway hyper-responsiveness, itch-scratch responses, infiltration and activation of eosinophils and neutrophils, vascular remodeling, and nociceptive flexor responses. LPA also induces histamine release from mouse and rat mast cells. In one aspect, the effects of LPA are mediated through LPA₁ and/or LPA₃. In one aspect, compounds of Formula (I) are used in the treatment of various allergic disorders in a mammal In one aspect, compounds of Formula (I) are used in the treatment of respiratory diseases, disorders or conditions in a mammal In one aspect, compounds of Formula (I) are used in the treatment of asthma in a mammal In one aspect, compounds of Formula (I) are used in the treatment of chronic asthma in a mammal.

The term “respiratory disease,” as used herein, refers to diseases affecting the organs that are involved in breathing, such as the nose, throat, larynx, eustachian tubes, trachea, bronchi, lungs, related muscles (e.g., diaphram and intercostals), and nerves. Respiratory diseases include, but are not limited to, asthma, adult respiratory distress syndrome and allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, acute severe asthma, chronic asthma, clinical asthma, nocturnal asthma, allergen-induced asthma, aspirin-sensitive asthma, exercise-induced asthma, isocapnic hyperventilation, child-onset asthma, adult-onset asthma, cough-variant asthma, occupational asthma, steroid-resistant asthma, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, including chronic bronchitis or emphysema, pulmonary hypertension, interstitial lung fibrosis and/or airway inflammation and cystic fibrosis, and hypoxia.

In one aspect, presented herein is the use of compounds of Formula (I) in the treatment or prevention of chronic obstructive pulmonary disease in a mammal comprising administering to the mammal at least once an effective amount of at least one compound of Formula (I). In addition, chronic obstructive pulmonary disease includes, but is not limited to, chronic bronchitis or emphysema, pulmonary hypertension, interstitial lung fibrosis and/or airway inflammation, and cystic fibrosis.

The nervous system is a major locus for LPA₁ expression. In one aspect, provided is a compound of Formula (I) for use in the treatment or prevention of a nervous system disorder in a mammal The term “nervous system disorder,” as used herein includes, but is not limited to, Alzheimer's Disease, cerebral edema, cerebral ischemia, stroke, multiple sclerosis, neuropathies, Parkinson's Disease, multiple sclerosis, retinal ischemia, post-surgical cognitive dysfunction, migraine, peripheral neuropathy/neuropathic pain, spinal cord injury, cerebral edema and head injury.

Angiogenesis, the formation of new capillary networks from pre-existing vasculature, is normally invoked in wound healing, tissue growth and myocardial angiogenesis after ischemic injury. Peptide growth factors and lysophospholipids control coordinated proliferation, migration, adhesion, differentiation and assembly of vascular endothelial cells (VECs) and surrounding vascular smooth-muscle cells (VSMCs). In one aspect, dysregulation of the processes mediating angiogenesis leads to atherosclerosis, hypertension, tumor growth, rheumatoid arthritis and diabetic retinopathy.

The specific effects of LPA are receptor-mediated.

In one aspect, compounds of Formula (I) are used to treat or prevent cardiovascular disease in mammal, including but not limited to: arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis, stroke; peripheral obstructive arteriopathy of a limb, an organ, or a tissue; reperfusion injury following ischemia of the brain, heart, kidney or other organ or tissue; endotoxic, surgical, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vasoconstriction (including that associated with migraines); vascular abnormality, inflammation, insufficiency limited to a single organ or tissue.

In one aspect, provided herein are methods for preventing or treating vasoconstriction, atherosclerosis and its sequelae myocardial ischemia, myocardial infarction, aortic aneurysm, vasculitis and stroke comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I) or pharmaceutical composition or medicament which includes a compound of Formula (I).

In one aspect, provided herein are methods for reducing cardiac reperfusion injury following myocardial ischemia and/or endotoxic shock comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, provided herein are methods for reducing the constriction of blood vessels in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, provided herein are methods for lowering or preventing an increase in blood pressure of a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

LPA is associated with various inflammatory/immune diseases. In one aspect, compounds of Formula (I) are used to treat or prevent inflammation in a mammal. In one aspect, antagonists of LPA₁ and/or LPA₃ find use in the treatment or prevention of inflammatory/immune disorders in a mammal.

Examples of inflammatory/immune disorders include psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, asthma, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, eczema, allogeneic or xenogeneic transplantation (organ, bone marrow, stem cells and other cells and tissues) graft rejection, graft-versus-host disease, lupus erythematosus, inflammatory disease, type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis and atopic dermatitis.

Other Diseases, Disorders or Conditions

In accordance with one aspect, are methods for treating, preventing, reversing, halting or slowing the progression of LPA-dependent or LPA-mediated diseases or conditions once it becomes clinically evident, or treating the symptoms associated with or related to LPA-dependent or LPA-mediated diseases or conditions, by administering to the mammal a compound of Formula (I). In certain embodiments, the subject already has a LPA-dependent or LPA-mediated disease or condition at the time of administration, or is at risk of developing a LPA-dependent or LPA-mediated disease or condition.

In certain aspects, are methods for preventing or treating eosinophil and/or basophil and/or dendritic cell and/or neutrophil and/or monocyte and/or T-cell recruitment comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In certain aspects, are methods for the treatment of cystitis, including, e.g., interstitial cystitis, comprising administering at least once to the mammal a therapeutically effective amount of at least one compound of Formula (I).

In accordance with one aspect, methods described herein include the diagnosis or determination of whether or not a patient is suffering from a LPA-dependent or LPA-mediated disease or condition by administering to the subject a therapeutically effective amount of a compound of Formula (I) and determining whether or not the patient responds to the treatment.

In one aspect provided herein are compounds of Formula (I), pharmaceutically acceptable salts, pharmaceutically acceptable prodrugs, and pharmaceutically acceptable solvates thereof, which are antagonists of at least one LPA receptor (e.g. LPA₁, LPA₂, LPA₃) and are used to treat patients suffering from one or more LPA-dependent or LPA-mediated conditions or diseases, including, but not limited to, lung fibrosis, kindney fibrosis, liver fibrosis, scarring, asthma, rhinitis, chronic obstructive pulmonary disease, pulmonary hypertension, interstitial lung fibrosis, arthritis, allergy, psoriasis, inflammatory bowel disease, adult respiratory distress syndrome, myocardial infarction, aneurysm, stroke, cancer, pain, proliferative disorders and inflammatory conditions. In some embodiments, LPA-dependent conditions or diseases include those wherein an absolute or relative excess of LPA is present and/or observed.

In any of the aforementioned aspects the LPA-dependent or LPA-mediated diseases or conditions include, but are not limited to, organ fibrosis, asthma, allergic disorders, chronic obstructive pulmonary disease, pulmonary hypertension, lung or pleural fibrosis, peritoneal fibrosis, arthritis, allergy, cancer, cardiovascular disease, ult respiratory distress syndrome, myocardial infarction, aneurysm, stroke, and cancer.

In one aspect, compounds of Formula (I) are used to improve the corneal sensitivity decrease caused by corneal operations such as laser-assisted in situ keratomileusis (LASIK) or cataract operation, corneal sensitivity decrease caused by corneal degeneration, and dry eye symptom caused thereby.

In one aspect, presented herein is the use of compounds of Formula (I) in the treatment or prevention of ocular inflammation and allergic conjunctivitis, vernal keratoconjunctivitis, and papillary conjunctivitis in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, presented herein is the use of compounds of Formula (I) in the treatment or prevention of Sjogren disease or inflammatory disease with dry eyes in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, LPA and LPA receptors (e.g. LPA₁) are involved in the pathogenesis of osteoarthritis. In one aspect, presented herein is the use of compounds of Formula (I) in the treatment or prevention of osteoarthritis in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, LPA receptors (e.g. LPA₁, LPA₃) contribute to the pathogenesis of rheumatoid arthritis. In one aspect, presented herein is the use of compounds of Formula (I) in the treatment or prevention of rheumatoid arthritis in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

In one aspect, LPA receptors (e.g. LPA₁) contribute to adipogenesis. In one aspect, presented herein is the use of compounds of Formula (I) in the promotion of adipose tissue formation in a mammal comprising administering at least once to the mammal an effective amount of at least one compound of Formula (I).

Compounds

In some embodiments, provided herein is a compound having the structure of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein

-   -   R¹ is —CO₂H, —CO₂R^(D), —CN, tetrazolyl, —C(═O)NH₂, —C(═O)NHR¹⁰,         —C(═O)NHSO₂R¹⁰ or —C(═O)NHCH₂CH₂SO₃H; R^(D) is H or C₁-C₄alkyl;     -   L¹ is absent or C₁-C₆alkylene;     -   R³ is H, C₁-C₄alkyl, C₃-C₆cycloalkyl, or C₁-C₄-fluoroalkyl;     -   R⁷ is H or C₁-C₄alkyl;     -   R⁸ is H, C₁-C₄alkyl, or C₁-C₄-fluoroalkyl;     -   R¹⁰ is a C₁-C₆alkyl, C₁-C₆-fluoroalkyl, C₃-C₆cycloalkyl, or a         substituted or unsubstituted phenyl;     -   each of R^(A), R^(B), and R^(C) are independently selected from         H, F, Cl, Br, I, —CN, —OH, C₁-C₄alkyl, C₁-C₄-fluoroalkyl,         C₁-C₄-fluoroalkoxy, C₁-C₄alkoxy, and C₁-C₄heteroalkyl;     -   m is 0, 1, or 2; n is 0, 1, or 2; p is 0, 1, or 2.

For any and all of the embodiments, substituents are selected from among from a subset of the listed alternatives. For example, in some embodiments, R¹ is —CO₂H, —CO₂R¹, —CN, tetrazolyl, or —C(═O)NHSO₂R¹⁰. For example, in some embodiments, R¹ is —CO₂H or —CO₂(R^(D)). In some embodiments, R^(D) is H, —CH₃, or —CH₂CH₃. In some embodiments, R¹ is —CO₂H. In some embodiments, R¹ is —C(═O)NHSO₂R¹⁰. In some embodiments, R¹ is a carboxylic acid bioisostere.

In some embodiments, R³ is C₁-C₄alkyl. In some embodiments, R³ is —CH₃.

In some embodiments, R⁷ is H.

In some embodiments, R⁸ is H, C₁-C₄alkyl, or C₁-C₄-fluoroalkyl. In some embodiments, R⁸ is H. In some embodiments, R⁸ is H or C₁-C₄alkyl. In some embodiments, R⁸ is H. In some embodiments, R⁸ is H, —CH₃, or —CF₃. In some embodiments, R⁸ is —CH₃. In some embodiments, R⁸ is —CH₂CH₃.

In some embodiments, R¹⁰ is a C₁-C₆alkyl or a substituted or unsubstituted phenyl. In some embodiments, R¹⁰ is a C₁-C₆alkyl. In some embodiments, R¹⁰ is a substituted or unsubstituted phenyl.

In some embodiments, each R^(A) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃. In some embodiments, each R^(A) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃. In some embodiments, each R^(A) is independently selected from H, F, Cl, —CH₃, —CF₃, and —OH. In some embodiments, each R^(A) is independently selected from H, F, Cl, —CH₃, and —OH. In some embodiments, each R^(A) is H.

In some embodiments, each R^(B) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃. In some embodiments, each R^(B) is independently selected from H, F, Cl, —CH₃, —CF₃, and —OH. In some embodiments, each R^(B) is independently selected from H, F, Cl, —CH₃, and —OH. In some embodiments, each R^(B) is H.

In some embodiments, each R^(C) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃. In some embodiments, each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃. In some embodiments, each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, and —OH. In some embodiments, each R^(C) is independently selected from H, F, Cl, and —OH. In some embodiments, each R^(C) is independently selected from H, F, and Cl. In some embodiments, each R^(C) is H.

In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0 or 1. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, R¹ is —CO₂H, —CO₂R^(D) or —C(═O)NHSO₂R¹⁰; R³ is C₁-C₄alkyl; R⁷ is H; R¹⁰ is a C₁-C₆alkyl or a substituted or unsubstituted phenyl; each R^(A) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; each R^(B) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; each R^(C) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0 or 1; p is 0 or 1.

In some embodiments, R¹ is —CO₂H or —CO₂R¹; R³ is —CH₃ or —CH₂CH₃; R⁸ is H, —CH₃ or —CF₃; R^(D) is H, —CH₃, or —CH₂CH₃.

In some embodiments, R¹ is —CO₂H; R³ is —CH₃; R⁸ is —CH₃; L¹ is absent, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, —C(CH₂CH₃)₂—, —CH₂CH(CH₃)—, or —CH₂C(CH₃)₂—. In some embodiments, R¹ is —CO₂H; R⁸ is —CH₃; L¹ is —CH₂—. In some embodiments, R¹ is —CO₂H; R⁸ is —CH₃; L¹ is —CH₂—; m is 0; n is 0 or 1; p is 0.

In some embodiments, the compound of Formula (I) has the following structure:

In some embodiments, L¹ is absent, —CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, or —C(CH₂CH₃)₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0; n is 0, 1, or 2; p is 0.

In some embodiments, L¹ is absent or —CH₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, and —OH; n is 0 or 1.

In some embodiments, the compound of Formula (I) has the following structure:

In some embodiments, L¹ is absent, —CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, or —C(CH₂CH₃)₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0; n is 0, 1, or 2; p is 0.

In some embodiments, L¹ is absent or —CH₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, and —OH; n is 0 or 1. In some embodiments, L¹ is —CH₂—.

In some embodiments, R¹ is —C(═O)NHSO₂R¹⁰; R³ is —CH₃ or —CH₂CH₃; R⁸ is H, —CH₃ or —CF₃; R¹⁰ is —CH₃, or —CH₂CH₃.

In some embodiments, L¹ is as described in Table 1 and/or Table 2.

In some embodiments,

as defined in Table 1.

In some embodiments,

as defined in Table 1.

In some embodiments,

In some embodiments,

In some embodiments, the compound of Formula (I) has a structure selected from:

Any combination of the groups described above for the various variables is contemplated herein. Throughout the specification, groups and substituents thereof are chosen by one skilled in the field to provide stable moieties and compounds.

In some embodiments, compounds of Formula (I) include, but are not limited to, those described in Table 1, Table 2, Table 3 and FIGS. 1 to 7.

TABLE 1

Cmpd # R^(A) B —L¹— R^(D) C R⁸ M + H* 1 H Phen-1,4- —CH₂— H 2-Trifluoromethyl- CH₃ 525 ylene phenyl 2 H Phen-1,4- —CH₂— H 3-Trifluoromethyl- CH₃ 525 ylene phenyl 3 H Phen-1,4- —CH₂— H 2,4-Dichloro-phenyl CH₃ 525 ylene 4 H Phen-1,4- —CH₂— H 2-Fluoro-phenyl CH₃ 475 ylene 5 H Phen-1,4- —CH₂— H 3-Bromo-phenyl CH₃ 535 ylene 6 H Phen-1,4- —CH₂— H 2-Methoxy-phenyl CH₃ 487 ylene 7 H 2-Methoxypen- —CH₂— H 2-Chloro-phenyl CH₃ 521 1,5-ylene 8 H Phen-1,4- — H 2-Chloro-phenyl CH₃ 477 ylene 9 H Phen-1,2- — H 2-Chloro-phenyl CH₃ 477 ylene 10 H Phen-1,2- —CH₂— H 2-Chloro-phenyl CH₃ 491 ylene 11 H Phen-1,4- —CH₂— H 2-Chloro-phenyl CH₃ 491 ylene 12 H Phen-1,3- —CH₂— H 2-Chloro-phenyl CH₃ 491 ylene 13 H Phen-1,4- —CH₂CH₃— H 2-Chloro-phenyl CH₃ 505 ylene 14 H 2-Fluorophen- —CH₂— H 2-Chloro-phenyl CH₃ 509 1,5-ylene 15 H 4-Fluorophen- —CH₂— H 2-Chloro-phenyl CH₃ 509 1,5-ylene 16 H Phen-1,4- —CH₂— —CH₃ 2-Chloro-phenyl CH₃ 505 ylene 17 H Phen-1,4- —CH(CH₃)— —CH₂CH₃ 2-Chloro-phenyl CH₃ 533 ylene 18 H Phen-1,4- —CH(CH₃)— H 2-Chloro-phenyl CH₃ 505 ylene 19 H Phen-1,4- —C(CH₃)₂— H 2-Chloro-phenyl CH₃ 519 ylene 20 H Phen-1,4- —CH(CH₃)— H 2-Chloro-phenyl CH₃ 489 ylene 21 H Phen-1,4- —CH₂CH₂CH₂— H 2-Chloro-phenyl CH₃ 519 ylene 22 H Phen-1,3- — H 2-Chloro-phenyl CH₃ 477 ylene 23 H Phen-1,4- —CH₂— H 2-Fluoro-4- CH₃ 509 ylene chloro-phenyl 24 H Phen-1,4- —CH₂— H 2-Fluoro- (R)—CH₃ 475 ylene phenyl 25 —CH₃ Phen-1,4- —CH₂— H 2-Fluoro- (R)—CH₃ 489 ylene phenyl 26 H Phen-1,4- —C(CH₃)₂— H 2-Fluoro- CH₃ 503 ylene phenyl 27 H Phen-1,4- —CH₂— H 2-Chloro-phenyl (R)—CH₃ 491 ylene 28 H Phen-1,4- —C(CH₃)₂— H 2-Chloro-phenyl (R)—CH₃ 519 ylene 29 H Phen-1,4- —C(CH₃)₂— H 2-Fluoro- (R)—CH₃ 503 ylene phenyl 30 H Phen-1,4- —CH(CH₃)— H 2-Fluoro- (R)—CH₃ 489 ylene phenyl 31 H Phen-1,4- —CH(CH₃)— H 2-Chloro-phenyl (R)—CH₃ 505 ylene 32 H Phen-1,4- —CH₂— H 2,6-Dichloro- CH₃ 525 ylene phenyl 33 —CH₃ Phen-1,4- —CH(CH₃)— H 2-Fluoro- (R)—CH₃ 503 ylene phenyl 34 H Phen-1,4- —CH₂— H 2-Fluoro- (S)—CH₃ 475 ylene phenyl 35 H Phen-1,4- —CH₂— H 2-Chloro-phenyl (S)—CH₃ 491 ylene 36 H Phen-1,4- —CH₂— H 2-Chloro-phenyl H 477 ylene 37 H Phen-1,4- —CH₂— H Phenyl (R)—CH₃ 457 ylene 38 H Phen-1,4- —CH₂— H 2,3-Difluoro- CH₃ 493 ylene phenyl 39 H Phen-1,4- —CH₂— H 2,4-Difluoro- CH₃ 493 ylene phenyl 40 H Phen-1,4- —CH₂— H 2-Fluoro-4- CH₃ 505 ylene methoxy-phenyl 41 H Phen-1,4- —CH₂— H 2,5-Difluoro- CH₃ 493 ylene phenyl 42 H Phen-1,4- —CH₂— H 2,6-Difluoro- CH₃ 493 ylene phenyl 43 H Phen-1,3- —CH₂— H Phenyl (R)—CH₃ 457 ylene 44 H Phen-1,4- — H Phenyl (R)—CH₃ 443 ylene 45 H Phen-1,2- —CH₂— H Phenyl (R)—CH₃ 457 ylene 46 H Phen-1,4- —CH₂— H 2-Methyl-phenyl (R)—CH₃ 471 ylene 47 H Phen-1,4- —CH(CH₃)— H 2-Chloro-phenyl (R)—CH₃ 505 ylene 48 H Phen-1,4- —CH(CH₃)— H 2-Chloro-phenyl (R)—CH₃ 505 ylene 49 H Phen-1,4- —CH₂— H 2-Chloro-phenyl (R)—CH₃ 525 ylene 50 H Phen-1,4- —CH(CH₂CH₃)— H 2-Chloro-phenyl (R)—CH₃ 519 ylene 51 Cl Phen-1,4- —CH₂— H 2-Chloro-phenyl (R)—CH₃ 526 ylene 52 F Phen-1,4- —CH₂— H 2-Chloro-phenyl (R)—CH₃ 509 ylene 53 H Phen-1,4- — H 2-Chloro-phenyl (R)—CH₃ 477 ylene 56 H Phen-1,4- —CH₂— H 3,5-Dibromo-phenyl (R)—CH₃ 615 ylene 57 H Phen-1,4- —CH₂— H Phenyl (S)—CH₃ 457 ylene 58 H Phen-1,4- —CH₂— H 3-Hydroxy-phenyl (R)—CH₃ 473 ylene 59 H Phen-1,4- —CH₂— H Phenyl —CH₃ 457 ylene 60 H Phen-1,4- —CH₂— H Phenyl-d5 —CD₃ 466 ylene *mass spectrometric data

TABLE 2

Cmpd M + # L¹ R¹ R^(C) R⁸ H* 61 —CH₂— —CN Cl (R)—CH₃ 472 62 —CH₂— —CN F (R)—CH₃ 456 63 —CH₂— 2H-Tetrazol-5-yl F (R)—CH₃ 499 64 —CH₂— 2H-Tetrazol-5-yl Cl (R)—CH₃ 515 65 —CH₂— —C(═NH)—NH₂ F (R)—CH₃ 473 66 —CH₂— —C(═NH)—NH—Ac F (R)—CH₃ 515 67 —CH₂— —C(═O)—NHCH₂CH₂SO₃H H (R)—CH₃ 564 *mass spectrometric data

TABLE 3

Cmpd # R¹ R^(C) R⁸ 67 —C(═O)NHSO₂Me H (R)—CH₃ 68 —C(═O)NHSO₂Me H H 69 —C(═O)NHSO₂Me 2-F (R)—CH₃ 70 —C(═O)NHSO₂Me 2-F H 71 —C(═O)NHSO₂Me 2-Cl (R)—CH₃ 72 —C(═O)NHSO₂Me 2-Cl H 73 —C(═O)NHSO₂Me 2-CH₃ (R)—CH₃ 74 —C(═O)NHSO₂Me 2-CH₃ H 75 —C(═O)NHSO₂Me 2-CF₃ (R)—CH₃ 76 —C(═O)NHSO₂Me 2-CF₃ H 77 —C(═O)NHSO₂Me 3-F (R)—CH₃ 78 —C(═O)NHSO₂Me 3-F H 79 —C(═O)NHSO₂Me 3-Cl (R)—CH₃ 80 —C(═O)NHSO₂Me 3-Cl H 81 —C(═O)NHSO₂Me 3-CH₃ (R)—CH₃ 82 —C(═O)NHSO₂Me 3-CH₃ H 83 —C(═O)NHSO₂Me 3-CF₃ (R)—CH₃ 84 —C(═O)NHSO₂Me 3-CF₃ H 85 —C(═O)NHSO₂Ph H (R)—CH₃ 86 —C(═O)NHSO₂Ph H H 87 —C(═O)NHSO₂Ph 2-F (R)—CH₃ 88 —C(═O)NHSO₂Ph 2-F H 89 —C(═O)NHSO₂Ph 2-Cl (R)—CH₃ 90 —C(═O)NHSO₂Ph 2-Cl H 91 —C(═O)NHSO₂Ph 2-CH₃ (R)—CH₃ 92 —C(═O)NHSO₂Ph 2-CH₃ H 93 —C(═O)NHSO₂Ph 2-CF₃ (R)—CH₃ 94 —C(═O)NHSO₂Ph 2-CF₃ H 95 —C(═O)NHSO₂Ph 3-F (R)—CH₃ 96 —C(═O)NHSO₂Ph 3-F H 97 —C(═O)NHSO₂Ph 3-Cl (R)—CH₃ 98 —C(═O)NHSO₂Ph 3-Cl H 99 —C(═O)NHSO₂Ph 3-CH₃ (R)—CH₃ 100 —C(═O)NHSO₂Ph 3-CH₃ H 101 —C(═O)NHSO₂Ph 3-CF₃ (R)—CH₃ 102 —C(═O)NHSO₂Ph 3-CF₃ H

Synthesis of Compounds

Compounds of Formula (I) described herein are synthesized using standard synthetic techniques or using methods known in the art in combination with methods described herein. In additions, solvents, temperatures and other reaction conditions presented herein may vary.

The starting material used for the synthesis of the compounds of Formula (I) are either synthesized or obtained from commercial sources, such as, but not limited to, Sigma-Aldrich, Fluka, Acros Organics, Alfa Aesar, and the like. General methods for the preparation of compounds can be modified by the use of appropriate reagents and conditions for the introduction of the various moieties found in the formulae as provided herein.

In some embodiments, the compounds of Formula (I) are prepared as outlined herein.

Further Forms of Compounds

In one aspect, compounds of Formula (I) possess one or more stereocenters and each stereocenter exists independently in either the R or S configuration. The compounds presented herein include all diastereomeric, and enantiomeric forms. Stereoisomers are obtained, if desired, by methods such as, stereoselective synthesis and/or the separation of stereoisomers by chiral chromatographic columns.

The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of Formula (I), as well as metabolites and active metabolites of these compounds having the same type of activity. In some situations, compounds may exist as tautomers. All tautomers are included within the scope of the compounds presented herein. In specific embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In other embodiments, the compounds described herein exist in unsolvated form.

In some embodiments, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

In some embodiments, sites on the aromatic ring portion of compounds of Formula (I) are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures will reduce, minimize or eliminate this metabolic pathway. In specific embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.

In another embodiment, the compounds described herein are labeled isotopically or by another other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

“Pharmaceutically acceptable,” as used herein, refers a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound of Formula (I) with acids. Pharmaceutically acceptable salts are also obtained by reacting a compound of Formula (I) with a base to form a salt.

Compounds described herein may be formed as, and/or used as, pharmaceutically acceptable salts. The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with a pharmaceutically acceptable inorganic acid (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like); or with an organic acid (e.g. acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, and the like); (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium), an alkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion. In some cases, compounds described herein may coordinate with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, compounds described herein may form salts with amino acids such as, but not limited to, arginine, lysine, and the like. Acceptable inorganic bases used to form salts with compounds that include an acidic proton, include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. In some embodiments, a sodium salt of the compound of Formula (I) is prepared.

It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.

Compounds described herein, such as compounds of Formula (I), may be in various forms, including but not limited to, amorphous forms, milled forms and nano-particulate forms. In addition, compounds described herein include crystalline forms, also known as polymorphs. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound.

Certain Terminology

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed. In this application, the use of “or” or “and” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

An “alkyl” refers to an aliphatic hydrocarbon. The alkyl may be saturated or unsaturated. The alkyl, whether saturated or unsaturated, is a branched alkyl or straight chain alkyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl, and the like.

The term “alkylene” refers to a divalent alkyl radical. Typical alkylene groups include, but are not limited to, —CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂C(CH₃)₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, and the like.

An “alkoxy” group refers to a (alkyl)O— group, where alkyl is as defined herein.

“Cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl.

The term “halo” or, alternatively, “halogen” or “halide” means fluoro, chloro, bromo or iodo.

The term “fluoroalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by a fluorine atom.

The term “heteroalkyl” refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. NH or Nalkyl), sulfur, or combinations thereof. In some embodiments, one aspect, heteroalkyl refers to an alkyl group in which one of the skeletal atoms of the alkyl is oxygen.

The term “optionally substituted” or “substituted” means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CO₂H, —CO₂alkyl, —C(═O)NH₂, —C(═O)NHalkyl, —C(═O)N(alkyl)₂, —S(═O)₂NH₂, —S(═O)₂NH(alkyl), —S(═O)₂N(alkyl)₂, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, —S-alkyl, or —S(═O)₂alkyl. In some embodiments, an optional substituent is selected from halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CF₃, —OCH₃, —OCH₂CH₃, and —OCF₃. In some embodiments, substituted groups are substituted with one or two of the preceding groups. In some embodiments, substituted groups are substituted with one of the preceding groups.

The term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

The term “modulate,” as used herein, means to interact with a target either directly or indirectly so as to alter the activity of the target, including, by way of example only, to enhance the activity of the target, to inhibit the activity of the target, to limit the activity of the target, or to extend the activity of the target.

The term “modulator,” as used herein, refers to a molecule that interacts with a target either directly or indirectly. The interactions include, but are not limited to, the interactions of an agonist, partial agonist, an inverse agonist and antagonist. In one embodiment, a modulator is an antagonist.

The term “agonist,” as used herein, refers to a molecule such as a compound, a drug, an enzyme activator or a hormone modulator that binds to a specific receptor and triggers a response in the cell. An agonist mimics the action of an endogenous ligand (such as LPA, prostaglandin, hormone or neurotransmitter) that binds to the same receptor.

The term “antagonist,” as used herein, refers to a molecule such as a compound, which diminishes, inhibits, or prevents the action of another molecule or the activity of a receptor site. Antagonists include, but are not limited to, competitive antagonists, non-competitive antagonists, uncompetitive antagonists, partial agonists and inverse agonists.

The term “LPA-dependent”, as used herein, refers to conditions or disorders that would not occur, or would not occur to the same extent, in the absence of LPA.

The term “LPA-mediated”, as used herein, refers to refers to conditions or disorders that might occur in the absence of LPA but can occur in the presence of LPA.

“Selectivity” for one LPA receptor versus other LPA receptors means that the compound has an IC₅₀ (Ca Flux assay) for the indicated LPA receptor that is at least 10-fold less than the IC₅₀ for other LPA receptors. In some embodiments, selectivity for one LPA receptor versus other LPA receptor means that the compound has an IC₅₀ for the indicated LPA receptor that is at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold, less than the IC₅₀ for other LPA receptors. For example, a selective LPA₁ receptor antagonist has an IC₅₀ that is at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold, less than the IC₅₀ for other LPA receptors (e.g. LPA₂, LPA₃).

The terms “co-administration” or the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

The term “pharmaceutical combination” as used herein, means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. a compound of Formula (I) and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. a compound of Formula (I) and a co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkey, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice guinea pigs, and the like. In one embodiment, the mammal is a human

The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating or ameliorating at least one symptom of a disease disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.

Pharmaceutical Compositions/Formulations and Routes of Administration

In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999), herein incorporated by reference for such disclosure.

A pharmaceutical composition, as used herein, refers to a mixture of a compound of Formula (I) with other chemical components (i.e. pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof. The pharmaceutical composition facilitates administration of the compound to an organism.

Pharmaceutical formulations described herein are administerable to a subject in a variety of ways by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

In some embodiments, the compounds of Formula (I) are administered orally.

In some embodiments, the compounds of Formula (I) are administered topically. In such embodiments, the compound of Formula (I) is formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, shampoos, scrubs, rubs, smears, medicated sticks, medicated bandages, balms, creams or ointments. In one aspect, the compounds of Formula (I) are administered topically to the skin.

In another aspect, the compounds of Formula (I) are administered by inhalation.

In another aspect, the compounds of Formula (I) are formulated for intranasal administration. Such formulations include nasal sprays, nasal mists, and the like.

In another aspect, the compounds of Formula (I) are formulated as eye drops.

In any of the aforementioned aspects are further embodiments in which the effective amount of the compound of Formula (I) is: (a) systemically administered to the mammal; and/or (b) administered orally to the mammal; and/or (c) intravenously administered to the mammal; and/or (d) administered by inhalation to the mammal; and/or (e) administered by nasal administration to the mammal; or and/or (f) administered by injection to the mammal; and/or (g) administered topically to the mammal; and/or (h) administered by ophthalmic administration; and/or (i) administered rectally to the mammal; and/or (j) adminstered non-systemically or locally to the mammal

In any of the aforementioned aspects are further embodiments comprising single administrations of the effective amount of the compound, including further embodiments in which (i) the compound is administered once; (ii) the compound is administered to the mammal multiple times over the span of one day; (iii) continually; or (iv) continuously.

In any of the aforementioned aspects are further embodiments comprising multiple administrations of the effective amount of the compound, including further embodiments in which (i) the compound is administered continuously or intermittently: as in a a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the compound is administered to the mammal every 8 hours; (iv) the compound is administered to the mammal every 12 hours; (v) the compound is administered to the mammal every 24 hours. In further or alternative embodiments, the method comprises a drug holiday, wherein the administration of the compound is temporarily suspended or the dose of the compound being administered is temporarily reduced; at the end of the drug holiday, dosing of the compound is resumed. In one embodiment, the length of the drug holiday varies from 2 days to 1 year.

In certain embodiments, a compound as described herein is administered in a local rather than systemic manner

In some embodiments, the compound described herein is administered topically. In some embodiments, the compound described herein is administered systemically.

In some embodiments, the pharmaceutical formulation is in the form of a tablet. In other embodiments, pharmaceutical formulations of the compounds of Formula (I) are in the form of a capsule.

In one aspect, liquid formulation dosage forms for oral administration are in the form of aqueous suspensions or solutions selected from the group including, but not limited to, aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

For administration by inhalation, a compound of Formula (I) is formulated for use as an aerosol, a mist or a powder.

For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

In some embodiments, compounds of Formula (I) are prepared as transdermal dosage forms.

In one aspect, a compound of Formula (I) is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.

In some embodiments, the compounds described herein may be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.

In some embodiments, the compounds of Formula (I) are formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas.

Methods of Dosing and Treatment Regimens

In one embodiment, the compounds of Formula (I) are used in the preparation of medicaments for the treatment of LPA-dependent or LPA-mediated diseases or conditions. In addition, a method for treating any of the diseases or conditions described herein in a subject in need of such treatment, involves administration of pharmaceutical compositions that include at least one compound of Formula (I) or a pharmaceutically acceptable salt, active metabolite, prodrug, or solvate thereof, in therapeutically effective amounts to said subject.

In certain embodiments, the compositions containing the compound(s) described herein are administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest at least one of the symptoms of the disease or condition. Amounts effective for this use depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation clinical trial.

In prophylactic applications, compositions containing the compounds described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition.

In certain embodiments, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).

Doses employed for adult human treatment are typically in the range of 0.01 mg-5000 mg per day or from about 1 mg to about 1000 mg per day. In one embodiment, the desired dose is conveniently presented in a single dose or in divided doses.

Patient Selection

In any of the aforementioned aspects involving the prevention or treatment of LPA-mediated diseases or conditions are further embodiments comprising identifying patients by screening for LPA receptor gene SNPs. Patients can be further selected based on increased LPA receptor expression in the tissue of interest. LPA receptor expression are determined by methods including, but not limited to, northern blotting, western blotting, quantitative PCR (qPCR), flow cytometry, autoradiography (using a small molecule radioligand or PET ligand). In some embodiments, patients are selected based on the concentration of serum or tissue LPA measured by mass spectrometry. In some embodiments, patients are selected based on a combination of the above markers (increased LPA concentrations and increased LPA receptor expression).

Combination Treatments

In certain instances, it is appropriate to administer at least one compound of Formula (I) in combination with another therapeutic agent.

In one specific embodiment, a compound of Formula (I) is co-administered with a second therapeutic agent, wherein the compound of Formula (I) and the second therapeutic agent modulate different aspects of the disease, disorder or condition being treated, thereby providing a greater overall benefit than administration of either therapeutic agent alone.

For combination therapies described herein, dosages of the co-administered compounds vary depending on the type of co-drug(s) employed, on the specific drug(s) employed, on the disease or condition being treated and so forth. In additional embodiments, when co-administered with one or more other therapeutic agents, the compound provided herein is administered either simultaneously with the one or more other therapeutic agents, or sequentially.

If administration is simultaneous, the multiple therapeutic agents are, by way of example only, provided in a single, unified form, or in multiple forms.

In another embodiment described herein, methods for treatment of proliferative disorders, including cancer, comprises administration to a mammal a compound of Formula (I) in combination with one or more anti-cancer agents and/or radiation therapy.

In one aspect, compounds of Formula (I) are to treat or reduce fibrosis in a mammal In one aspect, compounds of Formula (I) are administered in combination with one or more immunosuppresants. In some embodiments, a compound of Formula (I) is adminsitered with corticosteroids.

In yet another embodiment described herein, methods for treating LPA-dependent or LPA-mediated conditions or diseases, such as the therapy of respiratory disorders (e.g., pulmonary fibrosis, asthma, COPD, rhinitis), comprises administration to a patient compounds, pharmaceutical compositions, or medicaments described herein in combination with at least one agent used in the treatment of respiratory conditions.

In some embodiments, compounds of Formula (I) are administered to a patient in combination with anti-inflammatory agents.

In one embodiment, compounds of Formula (I) are administered to a patient in combination with inhaled corticosteroids.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Synthesis of Compounds Example 1 Synthesis of (R)-2′-chloro-alpha-methylbenzyl alcohol

Using the procedure of Meier et al (Tetrahedron, 1996, 52, 589; Method 3), 2′-chloroacetophenone (Aldrich) was reduced to give (R)-2′-chloro-alpha-methylbenzylalcohol in at least 80 e.e. % (HPLC analysis of the acetate derivative (made by reacting the benzyl alcohol with acetyl chloride and triethylamine in methylene chloride) using Chiralcel OD eluted with 99:1 Hexane:ethanol. R isomer retention time 4.3 minutes).

Example 2 Synthesis of (S)-2′-chloro-alpha-methylbenzyl alcohol

Using the procedure of Meier et al (Tetrahedron, 1996, 52, 589; Method 3), 2′-chloroacetophenone (Aldrich) was reduced to give (S)-2′-chloro-alpha-methylbenzylalcohol in at least 80 e.e. % (HPLC analysis of the acetate derivative (made by reacting the benzyl alcohol with acetyl chloride and triethylamine in methylene chloride) using Chiralcel OD eluted with 99:1 Hexane:ethanol. S isomer retention time 5.3 minutes)

Example 3 Synthesis of (R)-2′-fluoro-alpha-methylbenzyl alcohol

Using the procedure of Meier et al (Tetrahedron, 1996, 52, 589; Method 3), 2′-fluoroacetophenone (Aldrich) was reduced to give (R)-2′-fluoro-alpha-methylbenzylalcohol in at least 80 e.e. % (HPLC analysis of the acetate derivative (made by reacting the benzyl alcohol with acetyl chloride and triethylamine in methylene chloride) using Chiralcel OD eluted with 99.8:0.2 Hexane:ethanol. R isomer retention time 5.9 minutes).

Example 4 Synthesis of (S)-2′-fluoro-alpha-methylbenzyl alcohol

Using the procedure of Meier et al (Tetrahedron, 1996, 52, 589; Method 3), 2′-fluoroacetophenone (Aldrich) was reduced to give (S)-2′-fluoro-alpha-methylbenzylalcohol in at least 80 e.e. % (HPLC analysis of the acetate derivative (made by reacting the benzyl alcohol with acetyl chloride and triethylamine in methylene chloride) using Chiralcel OD eluted with 99.8:0.2 Hexane:Ethanol. S isomer retention time 6.7 minutes).

Example 5 Step 1: Synthesis of (3-Bromo-4-methoxy-phenyl)-acetic acid ethyl ester

To 3-bromo-4-methoxyphenylacetic acid (24 g, 97.9 mmol) in ethanol (240 mL) was added thionyl chloride (7.8 mL, 107.7 mmol), and the reaction was stirred at room temperature for 1 hour. Once no starting material was seen by analytical LCMS, the mixture was basified with saturated aqueous NaHCO₃ and extracted with dichloromethane. The combined organic layers were dried over MgSO₄, filtered, and concentrated to give the desired product.

Step 2: Synthesis of [4-Methoxy-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester

(3-Bromo-4-methoxy-phenyl)-acetic acid ethyl ester (27.4 g, 100.3 mmol), bis(pinacolato)diboron (25.47 g, 100.3 mmol), and potassium acetate (24.6 g, 250.8 mmol) were combined in 1,4-dioxane (250 mL) under N₂. The solution was purged with N₂, and then (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (4.10 g, 5.02 mmol) was added and the reaction was heated to 110° C. overnight. The mixture was filtered through Celite and partitioned between EtOAc and brine. The aqueous layer was separated and extracted twice with EtOAc, and the combined organic layers were dried and concentrated. The residue was purified by silica gel chromatography (20-60% EtOAc in hexanes) to give the title compound.

Example 6 Synthesis of 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid Step 1: 3-Methylamino-but-2-enoic acid methyl ester

To a solution of methyl acetoacetate (29.4 g, 253 mmol) in methanol (30 mL) was added methylamine (33 wt % in EtOH; 48 mL, 385 mmol) dropwise at room temperature. The reaction was stirred for 1 hour, and then concentrated and dried to give the title compound as a white crystalline solid.

Step 2: 2-(4-Bromo-benzoyl)-3-oxo-butyric acid methyl ester

To 3-methylamino-but-2-enoic acid methyl ester (5.0 g, 39.1 mmol) in THF (70 mL) was added pyridine (3.7 mL, 47 mmol) dropwise. The mixture was cooled to 0° C., and 4-bromobenzoyl chloride (8.55 g, 39.1 mmol) in THF (30 mL) was added dropwise. The reaction was stirred at room temperature overnight, and then water was added. The mixture was extracted with EtOAc, and the combined organic layers were washed with water, dried, filtered, and concentrated to give the title compound.

Step 3: 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester

To a mixture of 2-(4-bromo-benzoyl)-3-oxo-butyric acid methyl ester (11 g, 39 mmol) in acetic acid (50 mL) was added hydroxylamine hydrochloride (2.66 g, 39 mmol), and the reaction was stirred at 115° C. for 1 hours. After cooling, saturated aqueous NaHCO₃ was added to the mixture to adjust to pH 8. The solution was extracted with EtOAc, and the combined organic layers were washed with brine, dried, filtered, and concentrated to give the title compound.

Step 4: 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid

Lithium hydroxide (2 g, 48 mmol) was added to a solution of 5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester (39 mmol) in methanol (50 mL) and water (10 mL), and the reaction was stirred at 60° C. for 1 hour. Acidic work-up gave the title compound.

Example 7 Synthesis of [4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester and 4-((Ethoxycarbonyl)methyl)phenylboronic acid Step 1: [4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester

Ethyl 4-bromophenylacetate (12 g, 49 mmol), bis(pinacolato)diboron (12.4 g, 59 mmol), 1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (2 g, 2.4 mmol), and potassium acetate (9.6 g, 98 mmol) were combined in 1,4-dioxane (100 mL), and the reaction was heated to 80° C. for 6 hours. The mixture was worked-up to give the title compound.

Step 2: 4-((Ethoxycarbonyl)methyl)phenylboronic acid

[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl (0.460 g, 1.58 mmol), sodium periodate (0.746 g, 3.49 mmol), and ammonium acetate (0.309 g, 4.01 mmol) were combined in 1:1 acetone:water and stirred overnight at room temperature. Aqueous work-up provided the title compound.

Example 8 Synthesis of [3-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid methyl ester and 3-((Methoxycarbonyl)methyl)phenylboronic acid Step 1: [3-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid methyl ester

Prepared according to the procedure described in Example 7, Step 1 using methyl 2-(3-bromophenyl)acetate and bis(pinacolato)diboron.

Step 2: 3-((Methoxycarbonyl)methyl)phenylboronic acid

Prepared according to the procedure described in Example 7, Step 2 using [3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid methyl ester.

Example 9 Synthesis of (4′-{3-Methyl-4-[1-(2-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 1) Step 1: 5-(4′-Ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (2.0 g, 7.07 mmol), [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (2.46 g, 8.5 mmol), tetrakis(triphenylphosphine)palladium(0) (0.80 g, 0.70 mmol), and sodium bicarbonate (4.3 g, 52 mmol) were combined in 1,4-dioxane (50 mL) and water (10 mL), and the reaction was stirred overnight at 80° C. to give the title compound.

Step 2: (4′-{3-Methyl-4-[1-(2-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester

5-(4′-Ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid (0.150 g, 0.41 mmol), alpha-methyl-2-trifluoromethylbenzyl alcohol (0.094 g, 0.49 mmol), diphenylphosphoryl azide (0.11 mL, 0.49 mmol), and triethylamine (0.11 mL, 0.49 mmol) were combined in toluene (3 mL) and stirred at 80° C. for 2 hours. After cooling, the mixture was worked-up to give the title compound.

Step 3: (4′-{3-Methyl-4-[1-(2-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid

(4′-{3-Methyl-4-[1-(2-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester (0.41 mmol) was treated with aqueous lithium hydroxide in methanol at 60° C. for 30 minutes. The mixture was purified by preparative HPLC to give the title compound.

Example 10 Synthesis of (4′-{3-Methyl-4-[1-(3-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 2)

Following the procedure described in Example 9, Step 2,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and alpha-methyl-3-trifluoromethylbenzyl alcohol were reacted to provide (4′-{3-methyl-4-[1-(3-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 9, Step 3.

Example 11 Synthesis of (4′-{4-[1-(2,4-Dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 3)

Following the procedure described in Example 9, Step 2,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 2,4-dichloro-alpha-methylbenzyl alcohol were reacted to provide (4′-{4-[1-(2,4-Dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 9, Step 3.

Example 12 Synthesis of (4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 4)

Following the procedure described in Example 9, Step 2,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 2-fluoro-alpha-methylbenzyl alcohol were reacted to provide (4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 9, Step 3.

Example 13 Synthesis of (4′-{4-[1-(3-Bromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 5)

Following the procedure described in Example 9, Step 2,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 3-bromo-alpha-methylbenzyl alcohol were reacted to provide (4′-{4-[1-(3-bromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 9, Step 3.

Example 14 Synthesis of (4′-{4-[1-(2-Methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 6)

Following the procedure described in Example 9, Step 2,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2-methoxyphenyl)ethanol were reacted to provide (4′-{4-[1-(2-Methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 9, Step 3.

Example 15 Synthesis of (3-Bromo-4-fluoro-phenyl)-acetic acid ethyl ester

Step 1: 3-Bromo-4-fluorophenylacetic acid (2.35 g, 9.0 mmol) was treated with concentrated sulfuric acid (2 mL) in ethanol (100 mL) at reflux for 2 hours. Purification by silica gel chromatography provided the title compound.

Example 16 Synthesis of (5-Bromo-2-fluoro-phenyl)-acetic acid ethyl ester

Step 1: Prepared according to the procedure described in Example 15, Step 1 using the following starting material: 5-bromo-2-fluorophenylacetic acid.

Example 17 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-methoxy-biphenyl-3-yl)-acetic acid (Compound 7)

Step 1: To 5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (5.764 g, 20.4 mmol) in toluene (200 mL) was added triethylamine (3.13 mL, 22.4 mmol) and diphenylphosphoryl azide (4.42 mL, 20.4 mmol), and the mixture was stirred for 5 minutes at room temperature. 2-Chloro-alpha-methylbenzyl alcohol (3.2 g, 22.4 mol) was added, and the reaction was stirred at 80° C. for 2 hours. The mixture was diluted with EtOAc (200 mL) and extracted with EtOAc. The combined organic layers were washed twice with water and once with brine, and the combined aqueous layers were back-extracted with EtOAc. The combined organic layers were concentrated, and the residue was purified by silica gel chromatography to give [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-chloro-phenyl)-ethyl ester.

Step 2: To [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-chloro-phenyl)-ethyl ester (0.100 g, 0.23 mmol) in DME (5 mL) was added [4-methoxy-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (0.074 g, 0.23 mmol), followed by potassium carbonate (0.079 g, 0.58 mmol) and water (3 mL). The solution was purged with N₂ for 10 minutes, and then tetrakis(triphenylphosphine)palladium(0) (0.027 g, 0.02 mmol) was added. The reaction was stirred at 100° C. until no starting material was seen by analytical LCMS and thin layer chromatography (tlc), and then cooled to room temperature and diluted with EtOAc. The mixture was extracted with EtOAc, and the combined organic layers were washed with water and brine. The combined aqueous layers were back-extracted with EtOAc, and the combined organic layers were dried and concentrated. The residue was purified by silica gel chromatography to give (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-methoxy-biphenyl-3-yl)-acetic acid ethyl ester.

Step 3: To (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-methoxy-biphenyl-3-yl)-acetic acid ethyl ester (0.105 g, 0.20 mmol) in methanol (20 mL) was added 1N aqueous LiOH (5 mL, 5 mmol), and the reaction was stirred for 2 hours. The mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried and concentrated to give the title compound.

Example 18 Synthesis of {3-Methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-chloro-phenyl)-ethyl ester (3.48 g, 8.0 mmol), bis(pinacolato)diboron (2.24 g, 8.8 mmol), and potassium acetate (2.88 g, 32.0 mmol) were combined in 1,4-dioxane (200 mL) under N₂. The solution was purged with N₂ for 10 minutes, and then (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (0.652 g, 0.8 mmol) was added and the reaction was heated to 80° C. for 6 hours, and then cooled to room temperature and stirred overnight. The mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed twice with water and once with brine, and the combined aqueous layers were back-extracted with EtOAc. The combined organic layers were concentrated and the residue was purified by silica gel chromatography (0-100% EtOAc in hexanes) to give the title compound.

Example 19 Synthesis of 4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-carboxylic acid (Compound 8)

Step 1: {3-Methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester (0.100 g, 0.21 mmol), 4-bromobenzoic acid (0.042 g, 0.21 mmol), and potassium carbonate (0.073 g, 0.53 mmol) were combined in DME (10 mL) and water (5 mL). The solution was purged with N₂ for 10 minutes, and then (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (0.014 g, 0.02 mmol) was added. The reaction was stirred at 70° C. overnight, until no starting material was seen by analytical LCMS. The mixture was cooled to room temperature and quenched with water. The mixture was extracted with EtOAc, and the combined organic layers were dried and concentrated. The residue was purified by preparative HPLC to give the title compound.

Example 20 Synthesis of 4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-carboxylic acid (Compound 9)

Step 1: {3-Methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, methyl 2-bromobenzoate, and bis(triphenylphosphine)palladium(II) dichloride were reacted as described in Example 17, Step 2 to provide 4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-carboxylic acid methyl ester.

Step 2: To 4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-carboxylic acid methyl ester (0.2 μmol) in methanol was added 1N aqueous LiOH (2 mL) and stirred overnight at room temperature. The solution was stirred at 40° C. for 3 days. The mixture was purified by preparative HPLC to give the title compound.

Example 21 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-yl)-acetic acid (Compound 10)

Step 1: Prepared according to the procedure described in Example 19, Step 1 using {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester and 2-bromophenylacetic acid.

Example 22 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 11)

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, ethyl 4-bromophenylacetate, and (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) were reacted to provide (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 23 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-3-yl)-acetic acid (Compound 12)

3-Bromophenylacetic acid (0.31 mmol) was treated with concentrated sulfuric acid in ethanol at reflux for 2 hours to give (3-bromo-phenyl)-acetic acid ethyl ester.

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, (3-bromo-phenyl)-acetic acid ethyl ester, and (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) were reacted to provide (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-3-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 24 Synthesis of 3-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 13)

3-(4-Bromo-phenyl)-propionic acid ethyl ester was prepared according to the procedure described in Example 15, Step 1 using 3-(4-bromophenyl)propionic acid.

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, 3-(4-bromo-phenyl)-propionic acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride were reacted to provide 3-(4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 25 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-fluoro-biphenyl-3-yl)-acetic acid (Compound 14)

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, (3-bromo-4-fluoro-phenyl)-acetic acid ethyl ester, and (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) were reacted to provide (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-fluoro-biphenyl-3-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 26 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-4-fluoro-biphenyl-3-yl)-acetic acid (Compound 15)

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, (5-bromo-2-fluoro-phenyl)-acetic acid ethyl ester, and (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) were reacted to provide (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-4-fluoro-biphenyl-3-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 27 Synthesis of (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid methyl ester (Compound 16)

To (4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (0.050 g, 0.1 mmol) in benzene (17 mL) and methanol (2 mL) was added (trimethylsilyl)diazomethane (2M in hexanes; 0.1 mL, 0.2 mmol), and the reaction was stirred at room temperature for 5 minutes. Additional (trimethylsilyl)diazomethane (2M in hexanes; 0.25 mL, 0.5 mmol) was added, and the reaction was stirred until no starting material was seen by analytical LCMS. The mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed twice with water and once with brine, and then dried and concentrated. The residue was purified by silica gel chromatography (0-80% EtOAc in hexanes) to give the title compound.

Example 28 Synthesis of 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester (Compound 17)

Step 1: To ethyl 4-bromophenylacetate (2.137 g, 8.8 mmol) in THF (20 mL) at −78° C. was added lithium bis(trimethylsilyl)amide (1M in hexane; 11.4 mL, 11.4 mmol), and the solution was stirred for 1 hour at −78° C. Iodomethane (0.71 mL, 11.4 mmol) in THF (5 mL) was added, and the reaction was warmed to room temperature. The mixture was quenched with water and concentrated. The residue was dissolved in EtOAc and washed three times with water and once with brine. The organic layer was dried and concentrated, and the crude material was purified by silica gel chromatography (0-60% EtOAc in hexanes) to give 2-(4-bromo-phenyl)-propionic acid ethyl ester.

Step 2: Following the procedure described in Example 17, Step 2 the title compound was prepared using {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, 2-(4-bromo-phenyl)-propionic acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride; further purification by preparative HPLC was required.

Example 29 Synthesis of 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 18)

Prepared according to the procedure described in Example 17, Step 3 using 2-(4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester.

Example 30 Synthesis of 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 19)

To sodium hydride (0.859 g, 35.8 mmol) and iodomethane (2.23 mL, 35.8 mmol) in dimethylformamide (80 mL) at 0° C. was added ethyl 4-bromophenylacetate (2.17 g, 9.0 mmol) dropwise, and the reaction was warmed to room temperature and stirred for 4 hours. The mixture was quenched with water and 1N aqueous HCl (20 mL), and then extracted with 1:1 EtOAc:hexanes. The combined organic layers were washed five times with water and once with brine, and the combined aqueous layers were back-extracted with EtOAc. The combined organic layers were dried and concentrated, and the residue was purified by silica gel chromatography (0-20% EtOAc in hexanes). Further purification by preparative HPLC provided 2-(4-bromo-phenyl)-2-methyl-propionic acid ethyl ester.

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, 2-(4-bromo-phenyl)-2-methyl-propionic acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride were reacted to provide 2-(4′-{4-[1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester; further purification by preparative HPLC was required. The ester was hydrolyzed to the acid as described in Example 17, Step 3; purification by preparative HPLC was utilized.

Example 31 Synthesis of 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 20)

Step 1: 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (4 g, 14.2 mmol) was dissolved in toluene (150 mL). Triethylamine (2.187 mL, 15.6 mmol) was added, followed by diphenylphosphoryl azide (3.372 mL, 15.6 mmol), and the mixture was stirred for 10 minutes. 2-Fluoro-alpha-methylbenzyl alcohol (2 g, 15.6 mmol) was added, and the reaction was stirred at 80° C. overnight. The mixture was cooled to room temperature and concentrated, and the residue was partitioned between with EtOAc and water. The organic layer was washed 4 times with water and once with brine, and then dried, filtered, and concentrated, and the residue was purified by silica gel chromatography (dry load; 0-100% EtOAc in hexanes) to give [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-fluoro-phenyl)-ethyl ester.

Step 2: {3-Methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-fluoro-phenyl)-ethyl ester

Prepared according to the procedure described in Example 18 using [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-fluoro-phenyl)-ethyl ester and bis(pinacolato)diboron.

Step 3: 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester

Prepared according to the procedure described in Example 17, Step 2 using {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-fluoro-phenyl)-ethyl ester, 2-(4-bromo-phenyl)-propionic acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride; purification by preparative HPLC, followed by silica gel chromatography, was utilized.

Step 4: 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid

Prepared as described in Example 17, Step 3 using 2-(4′-{4-[1-(2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester.

Example 32 Synthesis of 4-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-butyric acid (Compound 21)

4-(4-Bromo-phenyl)-butyric acid ethyl ester was prepared according to the procedure described in Example 23 using 4-(4-bromophenyl)butanoic acid.

Following the procedure described in Example 17, Step 2, {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-chloro-phenyl)-ethyl ester, 4-(4-bromo-phenyl)-butyric acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride were reacted to provide 4-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-butyric acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 33 Synthesis of 4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-3-carboxylic acid (Compound 22)

Step 1: [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-chloro-phenyl)-ethyl ester (0.200 g, 0.46 mmol), 3-carboxyphenylboronic acid (0.092 g, 0.55 mmol), and potassium carbonate (0.190 g, 1.37 mmol) were combined in 2:1 DME:water (5 mL), and the solution was purged with N₂ for 10 minutes. Tetrakis(triphenylphosphine)palladium(0) (0.053 g, 0.05 mmol) was added, and the reaction was purged with N₂ for an additional 10 minutes, and then sealed and stirred at 80° C. overnight. The mixture was partitioned between EtOAc and water, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over MgSO₄, filtered, and concentrated, and the residue was purified by preparative HPLC to give the title compound.

Example 34 Synthesis of (4′-{4-[1-(4-Chloro-2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 23)

Step 1: 5-(4′-Ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 1-(4-chloro-2-fluorophenyl)ethanol were reacted as described in Example 31, Step 1 to provide (4′-{4-[1-(4-Chloro-2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester.

Step 2: Excess lithium hydroxide was added to a solution of (4′-{4-[1-(4-chloro-2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester (0.100 g, 0.19 mmol) in methanol and water, and the reaction was stirred at room temperature overnight. The mixture was acidified with 1N aqueous HCl and extracted with EtOAc, and the combined organic layers were dried, filtered, and concentrated. The residue was purified by preparative HPLC to give the title compound.

Example 35 Synthesis of (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 24)

Step 1: 2′-Fluoroacetophenone (25 g, 180 mmol) and (S)-(−)-2-methyl-CBS-oxazaborolidine (6.02 g, 22 mmol) were combined in THF (200 mL). Borane methyl sulfide complex (2M in THF; 59.4 mL, 119 mmol) was added over 20 minutes, and the reaction was stirred at room temperature for 30 minutes. Methanol was added, and the mixture was worked-up with dichloromethane and water. The organic layer was concentrated to give (R)-1-(2-fluoro-phenyl)-ethanol in 90.6% e.e.

Step 2: 5-(4′-Ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-(2-fluoro-phenyl)-ethanol were reacted as described in Example 31, Step 1 to provide (4′-{4-[(R)-1-(2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester.

Step 3: The title compound was prepared as described in Example 34, Step 2 using (4′-{4-[(R)-1-(2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester.

Example 36 Synthesis of (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-acetic acid (Compound 25) Step 1: 4-Bromo-3-methyl-benzoyl chloride

To 4-bromo-3-methylbenzoic acid (8.6 g, 40.0 mmol) in CH₂Cl₂ was added DMF (0.4 mL), followed by oxalyl chloride (3.7 mL, 42 mmol) slowly over 10 minutes by syringe. The reaction was stirred for 1 hour at room temperature, and then concentrated to give the title compound.

Step 2: 2-(4-Bromo-3-methyl-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester

To 4-bromo-3-methyl-benzoyl chloride (40.0 mmoml) in tertahydrofuran (300 mL) wad added 3-methylamino-but-2-enoic acid methyl ester (5.12 g, 40.0 mmol), followed by pyridine (3.2 mL, 40.0 mmol), and the reaction was stirred overnight at room temperature. Solid pyridine hydrochloride coated the flask, so the mixture was decanted, and the solid was washed three times with EtOAc. The combined solutions were concentrated and diluted with EtOAc (500 mL), and the mixture was washed three times with water. The aqueous layer was separated and back-extracted with EtOAc, and the combined organic layers were dried over MgSO₄, filtered, and concentrated to give the title compound.

Step 3: 5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester

2-(4-Bromo-3-methyl-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester (40.0 mmol) and hydroxylamine hydrochloride (2.9 g, 41 mmol) were combined in acetic acid (100 mL) and stirred at 60° C. overnight. The mixture was concentrated, and the residue was partitioned between EtOAc and water. Saturated aqueous NaHCO₃ was added to neutralize residual acetic acid, and the organic layer was separated and washed twice with water. The combined aqueous layers were back-extracted with EtOAc, and the combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel chromatography to give the title compound.

Step 4: 5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazole-4-carboxylic acid

5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester (7.26 g, 23.4 mmol) was hydrolyzed with 1N aqueous LiOH. Acidic work-up provided the title compound.

Step 5: [5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester

5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazole-4-carboxylic acid (1.78 g, 6.0 mmol) was dissolved in toluene (50 mL). Triethylamine (0.92 mL, 6.6 mmol) was added, followed by diphenylphosphoryl azide (1.43 mL, 6.6 mmol), and the mixture was stirred for 10 minutes. (R)-1-(2-Fluoro-phenyl)-ethanol (0.924 g, 6.6 mmol) was added, and the reaction was stirred at 85° C. for 3 hours, and then overnight at room temperature. The mixture was concentrated, and the residue was diluted with dichloromethane and purified by silica gel chromatography to give the title compound.

Step 6: (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-acetic acid ethyl ester

[5-(4-Bromo-3-methyl-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (0.350 g, 0.80 mmol), [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (0.292 g, 0.96 mmol), and sodium bicarbonate (0.235 g, 2.8 mmol) were combined in 2:1 DME:water (11 mL), and the solution was purged with N₂ for 5 minutes. Bis(triphenylphosphine)palladium(II) dichloride (0.030 g, 0.04 mmol) was added, and the reaction was stirred at 80° C. for 2 hour. After work-up, the crude material was purified by silica gel chromatography to give the title compound.

Step 7: (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-acetic acid

(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-acetic acid ethyl ester (0.282 g, 0.53 mmol) was hydrolyzed with lithium hydroxide in methanol and water, and the crude material was purified by preparative HPLC to give the title compound.

Example 37 Synthesis of 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 26) Step 1: 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester

Prepared as described in Example 36, Step 6 using {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid 1-(2-fluoro-phenyl)-ethyl ester and 2-(4-bromo-phenyl)-2-methyl-propionic acid ethyl ester; purification by preparative HPLC was utilized.

Step 2: 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid

2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester (0.51 mmol) in Methanol (50 mL) was treated with lithium hydroxide (excess) as well as 1N aqueous LiOH (excess, and the reaction was stirred at room temperature. Analytical LCMS indicated that starting material was still present, so the reaction was stirred at 80° C. overnight. The mixture was neutralized with 1N HCl (20 mL) and extracted with EtOAc. The combined organic layers were washed with water, dried, and concentrated, and the residue was purified by preparative HPLC to give the title compound.

Example 38 Synthesis of (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 27) Step 1: (R)-1-(2-Chloro-phenyl)-ethanol

2′-Chloroacetophenone (8.4 mL, 65 mmol) and (S)-(−)-2-methyl-CBS-oxazaborolidine (0.90 g, 0.32 mmol) were combined in THF (75 mL). Borane methyl sulfide complex (2M in THF; 21.5 mL, 43 mmol) was added over 20 minutes, and the reaction was stirred at room temperature for 30 minutes. MeOH was added, and the mixture was worked-up with CH₂Cl₂ and H₂O. The organic layer was concentrated to give the title compound.

Step 2: [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (10 g, 35 mmol), (R)-1-(2-chloro-phenyl)-ethanol (6.6 g, 42 mmol), triethylamine (10 mL, 70 mmol), and diphenylphosphoryl azide (11.5 g, 42 mmol) were combined in toluene (100 mL) and stirred at 90° C. for 1 hour. The mixture was concentrated, and the residue was purified by silica gel chromatography (0-30% EtOAc in hexanes). The isolated product was recrystallized in 5:1 hexanes:acetone to give the title compound in >99% e.e (by chiral HPLC. Chiracel OD 98.4% hexanes/1.6% Ethanol. Major isomer 27.9 min minor isomer 32.7 min)

Step 3: [4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester

Ethyl 4-bromophenylacetate (9.72 g, 40 mmol), bis(pinacolato)diboron (12.2 g, 48 mmol), and potassium acetate (18.8 g, 1690 mmol) were combined in 1,4-dioxane, and the mixture was purged with N₂ for 10 minutes. 1,1′-Bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (3.26 g, 4.0 mmol) was added, and the reaction was heated to 80° C. for 24 hours, and at room temperature for 2 days. The mixture was diluted with H₂O and extracted with EtOAc. The combined organic layers were washed with 4 times with H₂O, once with brine, and then dried, filtered, and concentrated. The residue was dissolved in CH₂Cl₂ and filtered through Celite to remove solids. The filtrate was concentrated, and the crude material was purified by silica gel chromatography (0-30% EtOAc in hexanes) to give the title compound.

Step 4: (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (3.0 g, 6.9 mmol), [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (2.2 g, 7.6 mmol), dichloro-bis(triphenylphosphine)palladium(II) (0.25 g, 0.35 mmol), and sodium bicarbonate (1.7 g, 20.7 mmol) were combined in DME (30 mL) and H₂O (10 mL), and the reaction was stirred at 80° C. for 2 hours. After aqueous work-up, the crude material was purified by silica gel chromatography (0-40% EtOAc in hexanes) to give the title compound.

Step 5: (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid

4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester (3 g, 5.9 mmol) in MeOH and H₂O was treated with lithium hydroxide (1 g, 23.8 mmol), and the reaction was stirred at 60° C. Acidic work-up provided the title compound.

Example 39 Synthesis of 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 28) Step 1: 2-Methyl-2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester

Prepared as described in Example 18 using 2-(4-bromo-phenyl)-2-methyl-propionic acid ethyl ester and bis(pinacolato)diboron.

Step 2: 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester

Prepared as described in Example 36, Step 6 using [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and 2-methyl-2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester.

Step 3: 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid

Prepared as described in Example 17, Step 3 using 2-(4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester.

Example 40 Synthesis of 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 29)

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester and 2-methyl-2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester were reacted to provide 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 41 Synthesis of 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 30)

2-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester was prepared according to the procedure described in Example 18 using 2-(4-bromo-phenyl)-propionic acid ethyl ester and bis(pinacolato)diboron.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester and 2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester were reacted to provide 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 42 Synthesis of 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 31)

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and 2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester were reacted to provide 2-(4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 43 Synthesis of [5-(4′-Cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Compound 61)

Prepared as described in Example 36, Step 6 using [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and 4-cyanomethylphenylboronic acid.

Example 44 Synthesis of [5-(4′-Cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 62)

Prepared as described in Example 36, Step 6 using [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester and 4-cyanomethylphenylboronic acid.

Example 45 Synthesis of {3-Methyl-5-[4′-(2H-tetrazol-5-ylmethyl)-biphenyl-4-yl]-isoxazol-4-yl}-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 63)

To a solution of [5-(4′-cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (0.200 g, 0.44 mmol) in toluene (5 mL) was added dibutyltin oxide (0.011 g, 0.04 mmol), followed by azidotrimethylsilane (0.07 mL, 0.53 mmol), and the reaction was stirred at 100° C. for 5 hours, and then stirred at room temperature overnight. The mixture was concentrated, and the residue was dissolved in EtOAc and washed with water and brine. The organic layer was dried and concentrated, and the crude material was purified by preparative HPLC to give the title compound.

Example 46 Synthesis of {3-Methyl-5-[4′-(2H-tetrazol-5-ylmethyl)-biphenyl-4-yl]-isoxazol-4-yl}-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Compound 64)

Prepared as described in Example 45 using [5-(4′-cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and azidotrimethylsilane.

Example 47 Synthesis of {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 37) Step 1: (R)-1-Phenyl-ethanol

To acetophenone (19.47 mL, 166.6 mmol) in THF (100 mL) was added (S)-(−)-2-methyl-CBS-oxazaborolidine (4.62 g, 16.6 mmol), and the reaction was cooled to 0° C. Borane methyl sulfide complex (2M in THF; 50 mL, 100 mmol) was added over 15 minutes, and the reaction was stirred at room temperature. Aqueous work-up gave the title compound.

Step 2: [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (25 g, 88.7 mmol) in toluene (500 mL) was added triethylamine (18.5 mL, 133 mmol), followed by diphenylphosphoryl azide (22.1 mL, 101.9 mmol). (R)-(+)-1-Phenylethyl alcohol (11.9 mL, 97.5 mmol) was added, and the reaction was stirred at 75° C. for 2 hours. The mixture was partitioned between EtOAc and H₂O and filtered through Celite. The aqueous layer was extracted with EtOAc, and the combined organic layers were dried over MgSO₄, filtered, and concentrated to give the title compound.

Step 3: {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester (39 g, 97.2 mmol), [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (31 g, 107 mmol), and sodium bicarbonate (32.6 g, 389 mmol) were combined in 3:1 DME:H₂O (500 mL), and the mixture was purged with N₂ for 15 minutes. (1,1′-Bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (2.13 g, 2.91 mmol) was added, and the reaction was purged with N₂ for an additional 10 minutes and then stirred at 90° C. overnight. The mixture was partitioned between EtOAc and H₂O, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with H₂O, dried over MgSO₄, filtered, and concentrated, and the residue was purified by silica gel chromatography (EtOAc/hexane gradient) to give the title compound.

Step 4: {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid

To a suspension of {4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester (24 g, 49 mmol) in 3:1 MeOH:H₂O (300 mL) was added lithium hydroxide (8.3 g, 198 mmol), and the reaction was stirred at room temperature overnight. The mixture was acidified and partitioned between EtOAc and H₂O. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with H₂O, dried over MgSO₄, filtered, and concentrated to give the title compound. Mass spec. data (M+H)=457.

Example 48 Synthesis of {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-3-yl}-acetic acid (Compound 43)

(3-Bromo-phenyl)-acetic acid ethyl ester and bis(pinacolato)diboron were reacted as described in Example 18 to provide [3-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester.

Following the procedure described in Example 17, Step 2, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester, [3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester, and bis(triphenylphosphine)palladium(II) dichloride were reacted to provide {4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-3-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 49 Synthesis of 4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-carboxylic acid (Compound 44)

Following the procedure described in Example 17, Step 2, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester and 4-ethoxycarbonylphenylboronic acid were reacted to provide 4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-carboxylic acid ethyl ester, which was hydrolyzed to the acid as described in Example 17, Step 3.

Example 50 Synthesis of (4′-{4-[1-(2,6-Dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 32)

Following the procedure described in Example 36, Step 5,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2,6-dichlorophenyl)ethanol were reacted to provide (4′-{4-[1-(2,6-dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 51 Synthesis of 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-propionic acid (Compound 33)

Following the procedure described in Example 36, Step 6, [5-(4-bromo-3-methyl-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester and 2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionic acid ethyl ester were reacted to provide 2-(4′-{4-[(R)-1-(2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-propionic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 52 Synthesis of (4′-{4-[(S)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 34)

(S)-1-(2-Fluoro-phenyl)-ethanol was prepared according to the procedure described in Example 35, Step 1 using 2′-fluoroacetophenone and (R)-(+)-2-methyl-CBS-oxazaborolidine.

Following the procedure described in Example 36, Step 5,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and (S)-1-(2-fluoro-phenyl)-ethanol were reacted to provide (4′-{4-[(S)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 53 Synthesis of (4′-{4-[(S)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 35)

(S)-1-(2-Chloro-phenyl)-ethanol was prepared as described in Example 35, Step 1 using 2′-chloroacetophenone and (R)-(+)-2-methyl-CBS-oxazaborolidine.

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (S)-1-(2-chloro-phenyl)-ethyl ester was prepared as described in Example 36, Step 5 using 5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and (S)-1-(2-chloro-phenyl)-ethanol.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (S)-1-(2-chloro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{-4-[(S)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 54 Synthesis of {4′-[4-(2-Chloro-benzyloxycarbonylamino)-3-methyl-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 36)

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 2-chloro-benzyl ester was prepared as described in Example 36, Step 5 using 5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and chlorobenzyl alcohol.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 2-chloro-benzyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide {4′-[4-(2-chloro-benzyloxycarbonylamino)-3-methyl-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 55 Synthesis of (4′-{4-[1-(2,3-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 38)

2′,3′-Difluoroacetophenone (0.337 g, 2.16 mmol) in methanol (15 mL) was treated with sodium borohydride (0.090 g, 2.37 mmol), and the reaction was stirred at room temperature for 30 minutes. The mixture was partitioned between dichloromethane and water, and the organic layer was separated, dried over MgSO₄, filtered, and concentrated to give 1-(2,3-difluoro-phenyl)-ethanol.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2,3-difluoro-phenyl)-ethanol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,3-difluoro-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,3-difluoro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[1-(2,3-difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 56 Synthesis of (4′-{4-[1-(2,4-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 39)

2′,4′-difluoroacetophenone was reduced to 1-(2,4-Difluorophenyl)ethanol as described in Example 55.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2,4-difluorophenyl)ethanol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,4-difluoro-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,4-difluoro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[1-(2,4-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 57 Synthesis of (4′-{4-[1-(2-Fluoro-4-methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 40)

2′-Fluoro-4′-methoxyacetophenone was reduced to 1-(2-fluoro-4-methoxyphenyl)ethan-1-ol as described in Example 55.

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2-fluoro-4-methoxyphenyl)ethan-1-ol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-fluoro-4-methoxy-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2-fluoro-4-methoxy-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[1-(2-fluoro-4-methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 58 Synthesis of (4′-{4-[1-(2,5-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 41)

2′,5′-Difluoroacetophenone was reduced to 1-(2,5-Difluorophenyl)ethanol as described in Example 55.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2,5-difluorophenyl)ethanol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,5-difluoro-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,5-difluoro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[1-(2,5-difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 59 Synthesis of (4′-{4-[1-(2,6-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 42)

2′,6′-difluoroacetophenone was reduced to 1-(2,6-difluorophenyl)ethan-1-ol as described in Example 55.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-(2,6-difluorophenyl)ethan-1-ol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,6-difluoro-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-(2,6-difluoro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[1-(2,6-difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 60 Synthesis of {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-2-yl}-acetic acid (Compound 45)

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester and [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-2-yl}-acetic ac id ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 61 Synthesis of {4′-[3-Methyl-4-((R)-1-o-tolyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 46)

2′-methylacetophenone and (S)-(−)-2-methyl-CBS-oxazaborolidine were reacted as described in Example 35, Step 1 to provide (R)-1-o-Tolyl-ethanol.

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-o-tolyl-ethanol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-o-tolyl-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-o-tolyl-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide {4′-[3-methyl-4-((R)-1-o-tolyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 62 Synthesis of 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Diastereomers—Compound 47 and Compound 48) Step 1: 2-(4-Bromo-phenyl)-propionic acid ethyl ester

2-(4-Bromo-phenyl)-propionic acid ethyl ester (10.0 g, 41 mmol) was dissolved in tetrahydrofuran (400 mL) and cooled to −78° C. Sodium bis(trimethylsilyl)amide (2M in tetrahydrofuran; 53.5 mL, 107 mmol) was added, and the mixture was stirred for 1 hour at −78° C. Iodomethane (2.814 mL, 45.1 mmol) was added, and the reaction was slowly warmed to room temperature and stirred overnight. The mixture was concentrated, and the residue was extracted with EtOAc. The combined organic layers were washed with water and brine, and then dried and concentrated. The crude material was purified by silica gel chromatography (0-15% EtOAc in hexanes) to give the title compound.

Step 2: 2-(4-Bromo-phenyl)-propionic acid

Prepared according to the procedure described in Example 6, Step 4 using 2-(4-bromo-phenyl)-propionic acid ethyl ester.

Step 3: 2-(4-Bromo-phenyl)-propionyl chloride

2-(4-Bromo-phenyl)-propionic acid (0.500 g, 2.18 mmol) in dichloromethane (20 mL) was treated with dimethylformamide (0.03 mL, 0.44 mmol), followed by oxalyl chloride (0.248 mL, 2.8 mmol) dropwise over 5 minutes, and the reaction was stirred at room temperature for 1 hour. The mixture was then concentrated and dried under vacuum to give the title compound.

Step 4: (4R,5S)-3-[2-(4-Bromo-phenyl)-propionyl]-4-methyl-5-phenyl-oxazolidin-2-one

(4R,5S)-(+)-4-Methyl-5-phenyl-2-oxazolidinone (0.348 g, 1.96 mmol) was dissolved in tetrahydrofuran (20 mL) and cooled to −78° C. n-Butyllithium (1.6M in hexanes; 1.64 mL, 2.62 mmol) was added, and the reaction was stirred for 1 hour at −78° C. 2-(4-Bromo-phenyl)-propionyl chloride (0.540 g, 2.18 mmol) in tetrahydrofuran (10 mL) was added, and the reaction was stirred for 30 minutes. The mixture was concentrated, and the residue was partitioned between EtOAc and water. The organic layer was separated, dried, and concentrated, and the crude material was purified by silica gel chromatography (0-25% EtOAc in hexanes) to give two diastereomers as separated products. Diastereomer A was the first product to come off the column, while Diastereomer B was the second product to come off the column.

Step 5: (4R,5S)-4-Methyl-5-phenyl-3-{2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionyl}-oxazolidin-2-one

Diastereomer A—Prepared as described in Example 5, Step 2 using (4R,5S)-3-[2-(4-bromo-phenyl)-propionyl]-4-methyl-5-phenyl-oxazolidin-2-one (Diastereomer A) and bis(pinacolato)diboron.

Diastereomer B—Prepared as described in Example 5, Step 2 using (4R,5S)-3-[2-(4-bromo-phenyl)-propionyl]-4-methyl-5-phenyl-oxazolidin-2-one (Diastereomer B) and bis(pinacolato)diboron.

Step 6: (3-Methyl-5-{4′-[1-methyl-2-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidin-3-yl)-2-oxo-ethyl]-biphenyl-4-yl}-isoxazol-4-yl)-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester

Diastereomer A—Prepared as described in Example 36, Step 6 using (4R,5S)-4-methyl-5-phenyl-3-{2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionyl}-oxazolidin-2-one (Diastereomer A) and [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester.

Diastereomer B—Prepared as described in Example 36, Step 6 using (4R,5S)-4-methyl-5-phenyl-3-{2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-propionyl}-oxazolidin-2-one (Diastereomer B) and [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester.

Step 7: 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid

Diastereomer A—Prepared as described in Example 34, Step 2 using (3-methyl-5-{4′-[1-methyl-2-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidin-3-yl)-2-oxo-ethyl]-biphenyl-4-yl}-isoxazol-4-yl)-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Diastereomer A).

Diastereomer B—Prepared as described in Example 34, Step 2 using (3-methyl-5-{4′-[1-methyl-2-((4R,5S)-4-methyl-2-oxo-5-phenyl-oxazolidin-3-yl)-2-oxo-ethyl]-biphenyl-4-yl}-isoxazol-4-yl)-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Diastereomer B)

Example 64 Synthesis of (3′-Chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 49)

Following the procedures described in Example 36: 4-bromo-2-chlorobenzoic acid and oxalyl chloride were reacted to provide 4-bromo-2-chloro-benzoyl chloride, which was reacted with 3-methylamino-but-2-enoic acid methyl ester to provide 2-(4-bromo-2-chloro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester. 2-(4-Bromo-2-chloro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester and hydroxylamine hydrochloride were then reacted to provide 5-(4-bromo-2-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester. Following Example 36, Step 4, 5-(4-bromo-2-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester was hydrolyzed to 5-(4-bromo-2-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid. 5-(4-Bromo-2-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-(2-chloro-phenyl)-ethanol were then reacted as described in Example 36, Step 5 to provide [5-(4-bromo-2-chloro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-2-chloro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (3′-chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 65 Synthesis of 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-butyric acid (Compound 50)

Prepared as described in Example 36, Step 6 using {3-methyl-5-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-isoxazol-4-yl}-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and 2-(4-bromophenyl)butanoic acid.

Example 66 Synthesis of (T-Chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 51)

Following the procedures described in Example 36: 4-bromo-3-chlorobenzoic acid and oxalyl chloride were reacted to provide 4-bromo-3-chloro-benzoyl chloride, which was then reacted with 3-methylamino-but-2-enoic acid methyl ester to provide 2-(4-bromo-3-chloro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester. 2-(4-Bromo-3-chloro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester and hydroxylamine hydrochloride were then reacted as described in Example 36, Step 3 to provide 5-(4-bromo-3-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester, which was then hydrolyzed to 5-(4-bromo-3-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid as described in Example 36, Step 4. 5-(4-Bromo-3-chloro-phenyl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-(2-chloro-phenyl)-ethanol were reacted to provide [5-(4-bromo-3-chloro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester as described in Example 36, Step 5.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-3-chloro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (2′-chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 67 Synthesis of (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-fluoro-biphenyl-4-yl)-acetic acid (Compound 52)

Following the procedures described in Example 36: 4-bromo-3-fluorobenzoyl chloride and oxalyl chloride were reacted to provide 4-bromo-3-fluoro-benzoyl chloride, which was then reacted with 3-methylamino-but-2-enoic acid methyl ester to provide 2-(4-bromo-3-fluoro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester. 2-(4-bromo-3-fluoro-benzoyl)-3-[(E)-methylimino]-butyric acid methyl ester and hydroxylamine hydrochloride were then reacted as described in Example 36, Step 3 to provide 5-(4-bromo-3-fluoro-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester, which was then hydrolyzed to 5-(4-bromo-3-fluoro-phenyl)-3-methyl-isoxazole-4-carboxylic acid as described in Example 36, Step 4. 5-(4-Bromo-3-fluoro-phenyl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-(2-chloro-phenyl)-ethanol were reacted to provide [5-(4-Bromo-3-fluoro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester as described in Example 36, Step 5.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-3-fluoro-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-fluoro-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 68 Synthesis of 4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-carboxylic acid (Compound 53)

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester and 4-ethoxycarbonylphenylboronic acid were reacted to provide 4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-carboxylic acid ethyl ester, which was hydrolyzed to the acid as described in Example 36, Step 7.

Example 71 Synthesis of (4′-{4-[(R)-1-(3,5-Dibromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 56)

To a solution of 3,5-dibromobenzoic acid (2.5 g, 8.9 mmol) in Et₂O (30 mL) at 0° C. was added methyl lithium (1.6M in diethyl ether; 12.3 mL, 19.6 mmol) was added dropwise. The reaction was warmed to room temperature and stirred for 2 hours. Acidic work-up, followed by silica gel chromatography, gave 1-(3,5-dibromo-phenyl)-ethanone.

1-(3,5-dibromo-phenyl)-ethanone and (S)-(−)-2-methyl-CBS-oxazaborolidine were reacted as described in Example 35, Step 1 to provide (R)-1-(3,5-dibromo-phenyl)-ethanol.

Following the procedure described in Example 36, Step 5,5-(4′-ethoxycarbonylmethyl-biphenyl-4-yl)-3-methyl-isoxazole-4-carboxylic acid and (R)-1-(3,5-dibromo-phenyl)-ethanol were reacted to provide (4′-{4-[(R)-1-(3,5-dibromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 72 Synthesis of {4′-[3-Methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 57)

2′-Chloroacetophenone and (R)-(+)-2-methyl-CBS-oxazaborolidine were reacted as described in Example 35, Step 1 to provide (S)-1-phenyl-ethanol.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and (S)-1-phenyl-ethanol were reacted as described in Example 36, Step 5 to provide [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (S)-1-phenyl-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (S)-1-phenyl-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide {4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 73 Synthesis of (4′-{4-[(R)-1-(3-Hydroxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 58) 3′-hydroxyacetophenone and (S)-(−)-2-methyl-CBS-oxazaborolidine were reacted as described in Example 35, Step 1 to provide 3-((R)-1-hydroxy-ethyl)-phenol.

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 3-((R)-1-hydroxy-ethyl)-phenol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(3-hydroxy-phenyl)-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(3-hydroxy-phenyl)-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide (4′-{4-[(R)-1-(3-hydroxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 74 Synthesis of {4′-[3-Methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 59)

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-phenylethanol were reacted as described in Example 36, Step 5 to provide [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-phenyl-ethyl ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-phenyl-ethyl ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester were reacted to provide {4′-[3-methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 75 Synthesis of {4′-[3-Methyl-4-(1-phenyl-ethoxy-d9-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 60)

5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and 1-phenylethanol-d₉ (fully dueterated 1-phenylethanol obtained from Carbocore) were reacted as described in Example 36, Step 5 to provide [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-phenyl-ethyl-d₉ ester.

Following the procedure described in Example 36, Step 6, [5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid 1-phenyl-ethyl-d₉ ester and [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-acetic acid ethyl were reacted to provide {4′-[3-methyl-4-(1-phenyl-ethoxy-d₉-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid ethyl ester, which was hydrolyzed to the acid as described in Example 34, Step 2.

Example 76 Synthesis of [5-(4′-Carbamimidoylmethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 65)

Step 1: [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester and 4-cyanomethylphenylboronic acid were reacted as described in Example 36, Step 6 to provide [5-(4′-cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester.

Step 2: [5-(4′-Cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (0.400 g, 0.88 mmol) in ethanol (5 mL) was treated with 4N HCl in 1,4-dioxane (5 mL), and the reaction was stirred overnight at room temperature. The mixture was concentrated to dryness, and then dissolved in 2M NH₃ in methanol. The reaction was stirred overnight at room temperature, and then additional 2M NH₃ in methanol was added and the reaction was stirred at 40° C. for 1.5 hours. The mixture was concentrated, and the residue was purified by preparative HPLC to give the title compound.

Example 77 Synthesis of {5-[4′-(2-Acetylamino-2-imino-ethyl)-biphenyl-4-yl]-3-methyl-isoxazol-4-yl}-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 66)

To [5-(4′-carbamimidoylmethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (0.055 g, 0.12 mmol) in dichloromethane (5 mL) was added diisopropylethylamine (0.052 mL, 0.3 mmol), followed by acetyl chloride (0.009 mL, 0.126 mmol), and the reaction was stirred at room temperature. Additional acetyl chloride (0.009 mL, 0.126 mmol) was added, and the reaction was stirred at room temperature. The mixture was quenched with water and extracted with dichloromethane. The combined organic layers were dried, filterd, and concentrated, and the residue was purified by preparative HPLC to give the title compound.

Example 78 Synthesis of [4′-(4-Amino-3-methyl-isoxazol-5-yl)-biphenyl-4-yl]acetic acid

[4′-(4-tert-Butoxycarbonylamino-3-methyl-isoxazol-5-yl)-biphenyl-4-yl]-acetic acid (1.0 mmol) was treated with trifluoroacetic acid (5 mL) for 1 hour. Work-up provided the title compound. Mass spec. data (M+H)=309.

Example 79 Synthesis of 2-(2-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetylamino)-ethanesulfonic acid (Compound 67)

Rats were dosed with {4′-[3-ethyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (30 mg/kg) and the bile was collected over 6 hours. The title compound was purified from bile by reverse phase HPLC. Mass spec. data (M+H)=564.

In some embodiments, Mass spectrometric data (mass spec. data) is obtained on with a Shimadzu LCMS 2010A.

In vitro Assay(s)

Example 80 Establishment of a CHO Cell Line Stably Expressing Human LPA₁

A 1.1 kb cDNA encoding the human LPA₁ receptor was cloned from human lung. Human lung RNA (Clontech Laboratories, Inc. USA) was reverse transcribed using the RETROscript kit (Ambion, Inc.) and the full-length cDNA for human LPA₁ was obtained by PCR of the reverse transcription reaction. The nucleotide sequence of the cloned human LPA₁ was determined by sequencing and confirmed to be identical to the published human LPA₁ sequence (An et al. Biochem. Biophys. Res. Commun. 231:619 (1997). The cDNA was cloned into the pcDNA5/FRT expression plasmid and transfected in CHO cells using lipofectamine 2000 (Invitrogen Corp., USA). Clones stably expressing human LPA₁ were selected using hygromycin and identified as cells that show Ca-influx in response to LPA.

Example 81 Generation of Cells Transiently Expressing Human LPA₂

A vector containing the human LPA₂ receptor cDNA was obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). The full-length cDNA fragment for human LPA₂ was obtained by PCR from the vector. The nucleotide sequence of the cloned human LPA₂ was determined by sequencing and confirmed to be identical to the published human LPA₂ sequence (NCBI accession number NM_(—)004720). The cDNA was cloned into the pcDNA3.1 expression plasmid and transfected into B103 cells (Invitrogen Corp., USA) by seeding cells in a 96-well poly-D-lysine coated plate at 30,000-35,000 cells per well together with 0.2 μl lipofectamine 2000 and 0.2 lag of the LPA₂ expression vector. Cells were cultured overnight in complete media before being assayed for LPA-induced Ca-influx.

Example 82 Establishment of a CHO Cell Line Stably Expressing Human LPA₃

A vector containing the human LPA₃ receptor cDNA was obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). The full-length cDNA fragment for human LPA₃ was obtained by PCR from the vector. The nucleotide sequence of the cloned human LPA₃ was determined by sequencing and confirmed to be identical to the published human LPA₃ sequence (NCBI accession number NM_(—)012152). The cDNA was cloned into the pcDNA5/FRT expression plasmid and transfected in CHO cells using lipofectamine 2000 (Invitrogen Corp., USA). Clones stably expressing human LPA₃ were selected using hygromycin and identified as cells that show Ca-influx in response to LPA.

Example 83 LPA1 and LPA3 Calcium Flux Assays

Human LPA₁ or LPA₃ expressing CHO cells are seeded at 20,000-45,000 cells per well in a 96-well poly-D-lysine coated plate one or two days before the assay. Prior to the assay, the cells are washed once with PBS and then cultured in serum-free media overnight. On the day of the assay, a calcium indicator dye (Calcium 4, Molecular Devices) in assay buffer (HBSS with Ca²⁺ and Mg²⁺ and containing 20 mM Hepes and 0.3% fatty-acid free human serum albumin) is added to each well and incubation continued for 1 hour at 37° C. 10 μl of test compounds in 2.5% DMSO are added to the cells and incubation continued at room temperature for 30 minutes. Cells are the stimulated by the addition of 10 nM LPA and intracellular Ca²⁺ measured using the Flexstation 3 (Molecular Devices). IC₅₀s are determined using Graphpad prism analysis of drug titration curves.

Example 84 LPA2 Calcium Flux Assay

Following an overnight culture with lipofectamine 2000 and the LPA₂ expression vector, the B103 cells are washed once with PBS then serum starved for 4 hours. A calcium indicator dye (Calcium 4, Molecular Devices) in assay buffer (HBSS with Ca²⁺ and Mg²⁺ and containing 20 mM Hepes and 0.3% fatty-acid free human serum albumin) is added to each well and incubation continued for 1 hour at 37° C. 10 μl of test compounds in 2.5% DMSO are added to the cells and incubation continued at room temperature for 30 minutes. Cells are the stimulated by the addition of 10 nM LPA and intracellular Ca²⁺ measured using the Flexstation 3 (Molecular Devices). IC₅₀s are determined using Graphpad prism analysis of drug titration curves.

Example 85 GTPγS Binding Assay

The ability of a compound to inhibit binding of GTP to LPA₁ is assessed via a membrane GTPγS assay. CHO cells stably expressing the recombinant human LPA₁ receptor are resuspended in 10 mM Hepes, 7.4 containing 1 mM DTT, lysed and centrifuged at 75,000×g to pellet the membranes. The membranes are resuspended in 10 mM Hepes, 7.4 containing 1 mM DTT and 10% glycerol. Membranes (−25 μg per well) are incubated in 96-well plates with 0.1 nM [³⁵S]-GTPγS, 900 nM LPA, 5 μM GDP, and test compound in Assay Buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 50 μg/ml saponin and 0.2% fatty-acid free human serum albumin) for 30 minutes at 30° C. The reactions are terminated by rapid filtration through Whatman GF/B glass fibre filter plates. The filter plates are washed 3 times with 1 ml cold Wash Buffer (50 mM Hepes, 7.5, 100 mM NaCl and 10 mM MgCl₂) and dried. Scintillant is then added to the plates and the radioactivity retained on the filters is determined on a Packard TopCount (Perkin Elmer). Specific binding is determined as total radioactive binding minus non-specific binding in the absence of the ligand (900 nM LPA). IC₅₀s were determined using Graphpad prism analysis of drug titration curves.

Illustrative in vitro biological data for representative compounds of Formula (I) is presented in the Table below. Unless otherwise noted, compounds that were tested had an IC₅₀ of less than 50 μM in the HLPA1 Ca Flux assay.

Compound HLPA1 Ca HLPA3 Ca Flux Number Flux IC50 (uM) IC50 (uM) 1 A C 2 B C 3 A C 4 A C 5 A C 6 A C 7 C C 8 A C 9 C ND 10 A C 11 A C 12 A B 13 A C 14 A C 15 A C 16 A C 17 A ND 18 A C 19 A ND 20 A C 21 A ND 22 A C 23 A C 24 A C 25 A C 26 A 27 A C 28 A B 29 A C 30 A C 31 A B 32 C ND 33 A ND 34 A C 35 B C 36 B ND 37 A C 38 A C 39 A C 40 C ND 41 A C 42 A D 43 B D 44 A C 45 C ND 46 A C 47 A B 48 A C 49 A C 50 A B 51 A C 52 A C 53 A B 56 C ND 57 A C 58 C D 61 A C 62 A D 63 A C 64 A C 65 C ND 66 A ND 67 A ND A = less than 0.3 μM; B = greater than 0.3 μM and less than 1 μM; C = greater than 1 μM and less than 50 μM; D = greater than 50 μM; ND = not determined.

Example 86 LPA1 Chemotaxis Assay

Chemotaxis of the A2058 human melanoma cells was measured using the Neuroprobe ChemoTx® System plates (8 μm pore size, 5.7 mm diameter sites). The filter sites were coated with 0.001% fibronectin (Sigma) in 20 mM Hepes, pH 7.4 and allowed to dry. A2058 cells were serum-starved for 24 hours, then harvested with Cell Stripper and resuspended in DMEM containing 0.1% fatty-acid-free bovine serum albumin (BSA) to a concentration of 1×10⁶/ml. Cells were mixed with an equal volume of test compound (2×) in DMEM containing 0.1% fatty-acid-free BSA and incubated at 37° C. for 15 minutes. LPA (100 nM in DMEM containing 0.1% fatty-acid-free BSA) or vehicle was added to each well of the lower chamber and 50 μl of the cell suspension/test compound mix was applied to the upper portion of the ChemoTx plate. Plates were incubated at 37° C. for three hours and then the cells removed from the upper portion by rinsing with PBS and scraping. The filter was dried then stained with HEMA 3 Staining System (Fisher Scientific). The absorbance of the filter was read at 590 nM and IC₅₀s were determined using Symyx Assay Explorer.

Compound 27 and Compound 37 inhibited LPA-driven chemotaxis (IC₅₀ less than 300 nM) of human A2058 melanoma cells.

In vivo Assay(s)

Example 87 Bleomycin-induced Lung Fibrosis Model in Mice

Female CD-1 mice (Harlan, 25-30 g) are housed 4 per cage, given free access to food and water and allowed to acclimate for at least 7 days prior to test initiation. After the habituation phase, mice are lightly anesthetized with isoflurane (5% in 100% O₂) and administered with bleomycin sulfate (0.01-5 U/kg, Henry Schein) via intratracheal instillation (Cuzzocrea S et al. Am J Physiol Lung Cell Mol. Physiol. 2007 May; 292(5):L1095-104. Epub 2007 Jan. 12.). Mice are returned to their cages and monitored daily for the duration of the experiment. Test compound or vehicle is delivered po, ip or sc daily. The route and frequency of dosing is based on previously determined pharmacokinetic properties. All animals are sacrificed using inhaled isoflurane 3, 7, 14, 21 or 28 days after bleomycin instillation. Following sacrifice, mice are intubated with a 20 gauge angiocatheter attached to a 1 ml syringe. Lungs are lavaged with saline to obtain bronchoalveolar lavage fluid (BALF) and then removed and fixed in 10% neutral buffered formalin for subsequent histopathological analysis. BALF is centrifuged for 10 min at 800×g to pellet the cells and the cell supernatant removed and frozen at −80° C. for subsequent protein analysis using the DC protein assay kit (Biorad, Hercules, Calif.) and soluble collagen analysis using Sircol (Biocolor Ltd, UK). BALF is analyzed for concentrations of inflammatory, pro-fibrotic and tissue injury biomarkers including transforming growth factor β1, hyaluronic acid, tissue inhibitor of metalloproteinase-1, matrix matelloproteinase-7, connective tissue growth factor and lactate dehydrogenase activity, using commercially available ELISA. The cell pellet is re-suspended in PBS. Total cell counts are then obtained using a Hemavet hematology system (Drew Scientific, Wayne, Pa.) and differential cells counts are determined using Shandon cytospin (Thermo Scientific, Waltham, Mass.). Lung tissue is stained using hematoxylin and eosin (H&E) and trichrome and lung fibrosis is determined by semiquantitative histopathological scoring (Ashcroft T. et al. J. Clin. Path. 1988; 41; 4, 467-470.) using light microscopy (10× magnification) and quantitative, computer-assisted densitometry of collagen in lung tissue sections using light microscopy. The data are plotted using Graphpad prism and statistical differences between groups determined.

In the acute setting (3 day), Compound 27 significantly reduced total protein, lactate dehydrogenase activity (LDH; tissue injury marker) and tissue inhibitor of metalloproteinase-1 (TIMP-1; pro-fibrotic marker) concentrations in broncheoalveolar lavage fluid (BALF). In the chronic setting (14 and 28 day) model, Compound 27 maintained mouse body weight and decreased inflammatory cell influx and fibrosis after a single bleomycin (1.5 units/kg) instillation and decreased pulmonary resistance and lung fibrosis following repeated bleomycin (3.0-5.0 units/kg/week) instillations.

Compound 37 reduced total protein, lactate and TIMP-1 in the BALF in the acute setting (3-day). Compound 37 decreased inflammatory cell influx and fibrosis after a single bleomycin instillation (3.0 units) in the chronic setting (14-days only).

Example 88 Mouse Carbon Tetrachloride (CCl₄)-induced Liver Fibrosis Model

Female C57BL/6 mice (Harlan, 20-25 g) housed 4/cage are given free access to food and water and allowed to acclimate for at least 7 days prior to test initiation. After the habituation phase, mice receive CCl₄ (0.5-1.0 ml/kg body weight) diluted in corn oil vehicle (100 μL volume) via i.p. injection twice a week for 4-6 weeks. (Higazi, A. A. et al., Clin Exp Immunol. 2008 April; 152(1):163-73. Epub 2008 Feb. 14.). Control mice receive an equivalent volume of corn oil vehicle only. Test compound or vehicle is delivered po, ip or sc daily. At the end of the study (4-6 weeks after first i.p. injection of CCl₄), mice are sacrificed using inhaled isoflurane and blood is drawn via cardiac puncture for subsequent analysis of ALT/AST levels. The liver is harvested, and one half of the liver is frozen at −80° C. and the other half is fixed in 10% neutral buffered formalin for histological assessment of liver fibrosis using light microscopy (10× magnification). Liver tissue homogenates are analyzed for collagen levels using Sircol (Biocolor Ltd, UK). Fixed Liver tissue is stained using hematoxylin and eosin (H&E) and trichrome and liver fibrosis is determined by quantitative, computer-assisted densitometry of collagen in liver tissue sections using light microscopy. Plasma and liver tissue lysates are also analyzed for concentrations of inflammatory, pro-fibrotic and tissue injury biomarkers including transforming growth factor β1, hyaluronic acid, tissue inhibitor of metalloproteinase-1, matrix matelloproteinase-7, connective tissue growth factor and lactate dehydrogenase activity, using commercially available ELISA. The resulting data are plotted using Graphpad prism and statistical differences between groups determined.

In this experiment, Compound 27 significantly reduced collagen deposition in the liver as compared to the untreated group. Compound 27 (30 mg/kg, po, qd) had the same effect on collagen deposition as pirfenidone. Compound 37 significantly reduced collagen deposition in the liver as compared to untreated control group.

Example 89 Mouse Intravenous LPA-induced Histamine Release

A mouse intravenous LPA-induced histamine release model is utilized to determine the in vivo potency of LPA₁ and LPA₃ receptor antagonists. Female CD-1 mice (weighing 25-35 grams) are administered compound (i.p., s.c. or p.o.) in a volume of 10 ml/kg 30 minutes to 24 hours prior to intravenous LPA challenge (300 μg/mouse in 0.1% FAF BSA) Immediately following LPA challenge mice are placed into an enclosed Plexiglas chamber and exposed to an isoflurane for a period of 2 minutes. They are removed, decapitated and trunk blood collected into tubes containing EDTA. Blood is then centrifuged at 10,000×g for 10 minutes at 4° C. Histamine concentrations in the plasma are determined by EIA. Drug concentrations in plasma are determined by mass spectrometry. The dose to achieve 50% inhibition of blood histamine release is calculated by nonlinear regression (Graphpad Prism) and plotted as the ED₅₀. The plasma concentration associated with this dose is plotted as the EC₅₀.

Example 90 Mouse Unilateral Ureteral Obstruction Kidney Fibrosis Model

Female C57BL/6 mice (Harlan, 20-25 g) housed 4/cage will be given free access to food and water and allowed to acclimate for at least 7 days prior to test initiation. After the habituation phase, mice undergo unilateral ureteral obstruction (UUO) surgery or sham to left kidney. Briefly, a longitudinal, upper left incision is performed to expose the left kidney. The renal artery is located and 6/0 silk thread is passed between the artery and the ureter. The thread is looped around the ureter and knotted 3 times insuring full ligation of ureter. The kidney is returned to abdomen, the abdominal muscle is sutured and the skin is stapled closed. Mice are returned to their cages and monitored daily for the duration of the experiment. Test compound or vehicle is delivered po, ip or sc daily. The route and frequency of dosing is based on previously determined pharmacokinetic properties. All animals are sacrificed using inhaled isoflurane 4, 8 or 14 days after UUO surgery. Following sacrifice blood is drawn via cardiac puncture, the kidneys are harvested and one half of the kidney is frozen at −80° C. and the other half is fixed in 10% neutral buffered formalin for histological assessment of kidney fibrosis using light microscopy (10× magnification). Kidney tissue homogenates are analyzed for collagen levels using Sircol (Biocolor Ltd, UK). Fixed kidney tissue is also stained using hematoxylin and eosin (H&E) and trichrome and kidney fibrosis is determined by quantitative, computer-assisted densitometry of collagen in liver tissue sections using light microscopy. Plasma and kidney tissue lysates are also analyzed for concentrations of inflammatory, pro-fibrotic and tissue injury biomarkers including transforming growth factor β1, hyaluronic acid, tissue inhibitor of metalloproteinase-1, matrix matelloproteinase-7, connective tissue growth factor and lactate dehydrogenase activity, using commercially available ELISA. The resulting data are plotted using Graphpad prism and statistical differences between groups determined

In this experiment, Compound 27 reduced kidney fibrosis by at least 20% as compared to the untreated group. Compound 37 reduced kidney fibrosis by 55% as compared to untreated group.

Example 91 Clinical Trial in Humans with Idiopathic Pulmonary Fibrosis (IPF) Purpose

The purposes of this study is to assess the efficacy of treatment with a compound of Formula (I) compared with placebo in patients with idiopathic pulmonary fibrosis (IPF) and to assess the safety of treatment with a compound of Formula (I) compared with placebo in patients with IPF.

The primary outcome variable is the absolute change in percent predicted forced vital capacity (FVC) from baseline to Week 72.

Secondary outcome measures include: composite outcomes of important IPF-related events; progression-free survival; categorical assessment of absolute change in percent predicted FVC from baseline to Week 72; change in Shortness-of-Breath from baseline to Week 72; change in percent predicted hemoglobin (Hb)-corrected carbon monoxide diffusing capacity (DLco) of the lungs from baseline to Week 72; change in oxygen saturation during the 6 minute walk test (6MWT) from baseline to Week 72; change in high-resolution computed tomography (HRCT) assessment from baseline to Week 72; change in distance walked in the 6MWT from baseline to Week 72.

Criteria

Patients eligible for this study include those patients that satisfy the following inclusion criteria: diagnosis of IPF; 40 to 80 years of age; FVC≧50% predicted value; DLco≧35% predicted value; either FVC or DLco≦90% predicted value; no improvement in past year; able to walk 150 meters in 6 minutes and maintain saturation≧83% while on no more than 6 L/min supplemental oxygen.

Patients are excluded from this study if they satisfy any of the following criteria: unable to undergo pulmonary function testing; evidence of significant obstructive lung disease or airway hyper-responsiveness; in the clinical opinion of the investigator, the patient is expected to need and be eligible for a lung transplant within 72 weeks of randomization; active infection; liver disease; cancer or other medical condition likely to result in death within 2 years; diabetes; pregnancy or lactation; substance abuse; personal or family history of long QT syndrome; other IPF treatment; unable to take study medication; withdrawal from other IPF trials. Patients are orally dosed with either placebo or an amount of compound of Formula (I) (1 mg/day-1000 mg/day). The primary outcome variable will be the absolute change in percent predicted FVC from Baseline to Week 72. Patients will receive blinded study treatment from the time of randomization until the last patient randomized has been treated for 72 weeks. A Data Monitoring Committee (DMC) will periodically review safety and efficacy data to ensure patient safety.

After week 72, patients who meet the Progression of Disease (POD) definition, which is a ≧10% absolute decrease in percent predicted FVC or a ≧15% absolute decrease in percent predicted DLco, will be eligible to receive permitted IPF therapies in addition to their blinded study drug. Permitted IPF therapies include corticosteroids, azathioprine, cyclophosphamide and N-acetyl-cysteine.

Example 92a Parenteral Pharmaceutical Composition

To prepare a parenteral pharmaceutical composition suitable for administration by injection (subcutaneous, intravenous, and the like), 100 mg of a water-soluble salt of a compound of Formula (I) is dissolved in sterile water and then mixed with 10 mL of 0.9% sterile saline. The mixture is incorporated into a dosage unit form suitable for administration by injection

In another embodiment, the following ingredients are mixed to form an injectable formulation: 1.2 g of a compound of Formulas (I), 2.0 mL of sodium acetate buffer solution (0.4 M), HCl (1 N) or NaOH (1 M) (q.s. to suitable pH), water (distilled, sterile) (q.s. to 20 mL). All of the above ingredients, except water, are combined and stirred and if necessary, with slight heating if necessary. A sufficient quantity of water is then added.

Example 92b Oral Pharmaceutical Composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of a compound of Formula (I) is mixed with 750 mg of starch. The mixture is incorporated into an oral dosage unit for, such as a hard gelatin capsule, which is suitable for oral administration.

Example 92c Sublingual (Hard Lozenge) Pharmaceutical Composition

To prepare a pharmaceutical composition for buccal delivery, such as a hard lozenge, mix 100 mg of a compound of Formula (I) with 420 mg of powdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42 mL mint extract. The mixture is gently blended and poured into a mold to form a lozenge suitable for buccal administration.

Example 92d Fast-Disintegrating Sublingual Tablet

A fast-disintegrating sublingual tablet is prepared by mixing 48.5% by weigh of a compound of Formula (I), 44.5% by weight of microcrystalline cellulose (KG-802), 5% by weight of low-substituted hydroxypropyl cellulose (50 μm), and 2% by weight of magnesium stearate. Tablets are prepared by direct compression (AAPS PharmSciTech. 2006; 7(2):E41). The total weight of the compressed tablets is maintained at 150 mg. The formulation is prepared by mixing the amount of compound of Formula (I) with the total quantity of microcrystalline cellulose (MCC) and two-thirds of the quantity of low-substituted hydroxypropyl cellulose (L-HPC) by using a three dimensional manual mixer (Inversina®, Bioengineering AG, Switzerland) for 4.5 minutes. All of the magnesium stearate (MS) and the remaining one-third of the quantity of L-HPC are added 30 seconds before the end of mixing.

Example 92e Inhalation Pharmaceutical Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mg of a compound of Formula (I) is mixed with 50 mg of anhydrous citric acid and 100 mL of 0.9% sodium chloride solution. The mixture is incorporated into an inhalation delivery unit, such as a nebulizer, which is suitable for inhalation administration.

In another embodiment, compound of Formula (I) (500 mg) is suspended in sterile water (100 mL), Span 85 (1 g) is added followed by addition of dextrose (5.5 g) and ascorbic acid (10 mg). Benzalkonium chloride (3 mL of a 1:750 aqueous solution) is added and the pH is adjusted to 7 with phosphate buffer. The suspension is packaged in sterile nebulizers.

Example 92f Rectal Gel Pharmaceutical Composition

To prepare a pharmaceutical composition for rectal delivery, 100 mg of a compound of Formula (I) is mixed with 2.5 g of methylcelluose (1500 mPa), 100 mg of methylparapen, 5 g of glycerin and 100 mL of purified water. The resulting gel mixture is then incorporated into rectal delivery units, such as syringes, which are suitable for rectal administration.

Example 92g Topical Gel Pharmaceutical Composition

To prepare a pharmaceutical topical gel composition, 100 mg of a compound of Formula (I) is mixed with 1.75 g of hydroxypropyl celluose, 10 mL of propylene glycol, 10 mL of isopropyl myristate and 100 mL of purified alcohol USP. The resulting gel mixture is then incorporated into containers, such as tubes, which are suitable for topicl administration.

Example 92h Ophthalmic Solution

To prepare a pharmaceutical opthalmic solution composition, 100 mg of a compound of Formula (I) is mixed with 0.9 g of NaCl in 100 mL of purified water and filterd using a 0.2 micron filter. The resulting isotonic solution is then incorporated into ophthalmic delivery units, such as eye drop containers, which are suitable for ophthalmic administration.

Example 92i Nasal Spray Solution

To prepare a pharmaceutical nasal spray solution, 10 g of a compound of Formula (I) is mixed with 30 mL of a 0.05M phosphate buffer solution (pH 4.4). The solution is placed in a nasal administrator designed to deliver 100 μl of spray for each application.

The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims. 

1-19. (canceled)
 20. A method for treating fibrosis in a mammal comprising administering a LPA₁ antagonist to the mammal in need thereof.
 21. The method of claim of claim 20, wherein the LPA₁ antagonist is a selective LPA₁ antagonist.
 22. The method of claim 20, wherein the fibrosis comprises fibrosis of organs or tissues in the mammal.
 23. The method of claim 22, wherein the fibrosis comprises fibrosis of the heart, kidney, liver, joints, lung, pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletal or digestive tract.
 24. The method of claim 20, wherein the fibrosis is lung fibrosis, kidney fibrosis, liver fibrosis, skin fibrosis, fibrosis of the gut, scleroderma, ocular fibrosis, spinal cord fibrosis, head and neck fibrosis, or myelofibrosis.
 25. The method of claim 20, wherein the fibrosis is lung fibrosis, kidney fibrosis, or liver fibrosis.
 26. The method of claim 20, wherein the fibrosis is idiopathic pulmonary fibrosis.
 27. The method of claim 20, wherein the fibrosis is scleroderma.
 28. The method of claim 20, wherein the fibrosis is ocular fibrosis.
 29. The method of claim 20, wherein the fibrosis is not responsive to treatment with pirfenidone.
 30. The method of claim 20, wherein the LPA₁ antagonist is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein, R¹ is —CO₂H, —CO₂R^(D), —CN, tetrazolyl, —C(═O)NH₂, —C(═O)NHR¹⁰, —C(═O)NHSO₂R¹⁰ or —C(═O)NHCH₂CH₂SO₃H; R^(D) is H or C₁-C₄alkyl; L¹ is absent or C₁-C₆alkylene; R³ is H, C₁-C₄alkyl, C₃-C₆cycloalkyl, or C₁-C₄-fluoroalkyl; R⁷ is H or C₁-C₄alkyl; R⁸ is H, C₁-C₄alkyl, or C₁-C₄-fluoroalkyl; R¹⁰ is a C₁-C₆alkyl, C₁-C₆-fluoroalkyl, C₃-C₆cycloalkyl, or a substituted or unsubstituted phenyl; each of R^(A), R^(B), and R^(C) are independently selected from H, F, Cl, Br, I, —CN, —OH, C₁-C₄alkyl, C₁-C₄-fluoroalkyl, C₁-C₄-fluoroalkoxy, C₁-C₄alkoxy, and C₁-C₄heteroalkyl; m is 0, 1, or 2; n is 0, 1, or 2; and p is 0, 1, or
 2. 31. The method of claim 30, wherein: R¹ is —CO₂H, —CO₂R^(D) or —C(═O)NHSO₂R¹⁰; R³ is C₁-C₄alkyl; R⁷ is H; R¹⁰ is a C₁-C₆alkyl or a substituted or unsubstituted phenyl; each R^(A) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; each R^(B) is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; each Rc is independently selected from H, F, Cl, Br, I, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0 or 1; and p is 0 or
 1. 32. The method of claim 31, wherein: R¹ is —CO₂H; L¹ is absent, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, —C(CH₂CH₃)₂—, —CH₂CH(CH₃)—, or —CH₂C(CH₃)₂—; R³ is —CH₃; R⁸ is H, —CH₃ or —CF₃.
 33. The method of claim 32, wherein the compound of Formula (I) has the following structure:


34. The method of claim 33, wherein: L¹ is absent, —CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, or —C(CH₂CH₃)₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0; n is 0, 1, or 2; and p is
 0. 35. The method of claim 32, wherein the compound of Formula (I) has the following structure:


36. The method of claim 35, wherein: L¹ is absent, —CH₂—, —CH(CH₃)—, —C(CH₃)₂—, —CH(CH₂CH₃)—, or —C(CH₂CH₃)₂—; each R^(C) is independently selected from H, F, Cl, —CH₃, —CF₃, —OH, —OCF₃, and —OCH₃; m is 0; n is 0, 1, or 2; and p is
 0. 37. The method of claim 31, wherein: R¹ is —C(═O)NHSO₂R¹⁰; R³ is —CH₃ or —CH₂CH₃; R⁸ is H, —CH₃ or —CF₃; and R¹⁰ is —CH₃, or —CH₂CH₃.
 38. The method of claim 30, wherein the compound of Formula (I) has the following structure:

wherein, R¹ is —CO₂H or —C(═O)NHSO₂R¹⁰; R⁸ is H, or —CH₃; R¹⁰ is a C₁-C₆alkyl or a substituted or unsubstituted phenyl; R^(C) is H, F, Cl, Br, I, —CN, —OH, C₁-C₄alkyl, C₁-C₄-fluoroalkyl, C₁-C₄-fluoroalkoxy, C₁-C₄alkoxy, or C₁-C₄heteroalkyl.
 39. The method of claim 20, wherein the LPA₁ antagonist is: (4′-{3-Methyl-4-[1-(2-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 1); (4′-{3-Methyl-4-[1-(3-trifluoromethyl-phenyl)-ethoxycarbonylamino]-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 2); (4′-{4-[1-(2,4-Dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 3); (4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 4); (4′-{4-[1-(3-Bromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 5); (4′-{4-[1-(2-Methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 6); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-methoxy-biphenyl-3-yl)-acetic acid (Compound 7); 4′-{-4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-carboxylic acid (Compound 8); 4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-carboxylic acid (Compound 9); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-2-yl)-acetic acid (Compound 10); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 11); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-3-yl)-acetic acid (Compound 12); 3-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 13); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-6-fluoro-biphenyl-3-yl)-acetic acid (Compound 14); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-4-fluoro-biphenyl-3-yl)-acetic acid (Compound 15); (4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid methyl ester (Compound 16); 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid ethyl ester (Compound 17); 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 18); 2-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 19); 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 20); 4-(4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-butyric acid (Compound 21); 4′-{4-[1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-3-carboxylic acid (Compound 22); (4′-{4-[1-(4-Chloro-2-fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 23); (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 24); (4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-acetic acid (Compound 25); 2-(4′-{4-[1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 26); (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 27); 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 28); 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-2-methyl-propionic acid (Compound 29); 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 30); 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 31); (4′-{4-[1-(2,6-Dichloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 32); 2-(4′-{4-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-methyl-biphenyl-4-yl)-propionic acid (Compound 33); (4′-{4-[(S)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 34); (4′-{4-[(S)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 35); {4′-[4-(2-Chloro-benzyloxycarbonylamino)-3-methyl-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 36); {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 37); (4′-{4-[1-(2,3-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 38); (4′-{4-[1-(2,4-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 39); (4′-{4-[1-(2-Fluoro-4-methoxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 40); (4′-{4-[1-(2,5-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 41); (4′-{4-[1-(2,6-Difluoro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 42); {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-3-yl}-acetic acid (Compound 43); 4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-carboxylic acid (Compound 44); {4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-2-yl}-acetic acid (Compound 45); {4′-[3-Methyl-4-((R)-1-o-tolyl-ethoxycarbonylamino)-isoxazol-5-yl}-biphenyl-4-yl]-acetic acid (Compound 46); 2-(4′-{4-[(R,R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 47); 2-(4′-{4-[(R,S)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-propionic acid (Compound 48); (3′-Chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 49); 2-(4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-butyric acid (Compound 50); (2′-Chloro-4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 51); (4′-{4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-2′-fluoro-biphenyl-4-yl)-acetic acid (Compound 52); 4′-{-4-[(R)-1-(2-Chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-carboxylic acid (Compound 53); (4′-{4-[(R)-1-(3,5-Dibromo-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 56); {4′-[3-Methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 57); (4′-{4-[(R)-1-(3-Hydroxy-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (Compound 58); {4′-[3-Methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetic acid (Compound 59); [5-(4′-Cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Compound 61); [5-(4′-Cyanomethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 62); {3-Methyl-5-[4′-(2H-tetrazol-5-ylmethyl)-biphenyl-4-yl]-isoxazol-4-yl}-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 63); {3-Methyl-5-[4′-(2H-tetrazol-5-ylmethyl)-biphenyl-4-yl]-isoxazol-4-yl}-carbamic acid (R)-1-(2-chloro-phenyl)-ethyl ester (Compound 64); [5-(4′-Carbamimidoylmethyl-biphenyl-4-yl)-3-methyl-isoxazol-4-yl]-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 65); {5-[4′-(2-Acetylamino-2-imino-ethyl)-biphenyl-4-yl]-3-methyl-isoxazol-4-yl}-carbamic acid (R)-1-(2-fluoro-phenyl)-ethyl ester (Compound 66); or 2-(2-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetylamino)-ethanesulfonic acid (Compound 67), or a pharmaceutically acceptable salt thereof. 